Streptococcus pyogenes antigens

ABSTRACT

The present invention relates to antigens, more particularly an antigen of  Streptococcus pygenes  (also called group A Streptococcus (GAS)) bacterial pathogen which is useful as vaccine component for therapy and/or prophylaxis.

FIELD OF THE INVENTION

[0001] The present invention is related to antigens, more particularly apolypeptide antigen of Streptococcus pyogenes (also called group AStreptococcus (GAS)) bacterial pathogen which may be useful forprophylaxis, diagnostic and/or therapy of streptococcal infection.

BACKGROUND OF THE INVENTION

[0002] Streptococci are gram (+) bacteria which are differentiated bygroup specific carbohydrate antigens A through O which are found at thecell surface. Streptococcus pyogenes isolates are further distinguishedby type-specific M protein antigens. M proteins are important virulencefactors which are highly variable both in molecular weights and insequences. Indeed, more than 80-M protein types have been identified onthe basis of antigenic differences.

[0003]Streptococcus pyogenes is responsible for many diverse infectiontypes, including pharyngitis, erysipelas and impetigo, scarlet fever,and invasive diseases such as bacteremia and necrotizing fasciitis andalso toxic shock. A resurgence of invasive disease in recent years hasbeen documented in many countries, including those in North America andEurope. Although the organism is sensitive to antibiotics, the highattack rate and rapid onset of sepsis results in high morbidity andmortality.

[0004] To develop a vaccine that will protect individuals fromStreptococcus pyogenes infection, efforts have concentrated on virulencefactors such as the type-specific M proteins. However, theamino-terminal portion of M proteins was found to induce cross-reactiveantibodies which reacted with human myocardium, tropomyosin, myosin, andvimentin, which might be implicated in autoimmune diseases. Others haveused recombinant techniques to produce complex hybrid proteinscontaining amino-terminal peptides of M proteins from differentserotypes. However, a safe vaccine containing all Streptococcus pyogenesserotypes will be highly complex to produce and standardize.

[0005] In addition to the serotype-specific antigens, otherStreptococcus pyogenes proteins have generated interest as potentialvaccine candidates. The C5a peptidase, which is expressed by at leastStreptococcus pyogenes 40 serotypes, was shown to be immunogenic inmice, but its capacity to reduce the level of nasopharyngealcolonization was limited. Other investigators have also focused on thestreptococcal pyrogenic exotoxins which appear to play an important rolein pathogenesis of infection. Immunization with these proteins preventedthe deadly symptoms of toxic shock, but did not prevent colonization.

[0006] Therefore there remains an unmet need for Streptococcus pyogenesantigens that may be used vaccine components for prophylaxis, diagnosticand/or therapy of Streptococcus infection.

SUMMARY OF THE INVENTION

[0007] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 70%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analogues orderivatives thereof.

[0008] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 95%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analogues orderivatives thereof.

[0009] In other aspects, there are provided novel polypeptides encodedby polynucleotides of the invention, vectors comprising polynucleotidesof the invention operably linked to an expression control region, aswell as host cells transfected with said vectors, pharmaceutical orvaccine compositions and methods of producing polypeptides comprisingculturing said host cells under conditions suitable for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is the DNA sequence of BVH-P1 gene from serotype 3 S.pyogenes strain ATCC12384 with a secretion signal at position 1 to 75;SEQ ID NO:1.

[0011]FIG. 2 is the amino acid sequence BVH-P1 protein from serotype 3S. pyogenes strain ATCC12384 with a secretion signal at position 1 to25; SEQ ID NO:2.

[0012]FIG. 3 is the DNA sequence of BVH-P1 gene from S. pyogenes strainLSPQ2699(ATCC19615) with a secretion signal at position 1 to 75; SEQ IDNO:3.

[0013]FIG. 4 is the amino acid sequence BVH-P1 protein from S. pyogenesstrain LSPQ2699(ATCC19615) with a secretion signal at position 1 to 25;SEQ ID NO:4.

[0014]FIG. 5 is the DNA sequence of BVH-P1 gene from S. pyogenes strainSPY57 with a secretion signal at position 1 to 75; SEQ ID NO:5. FIG. 6is the amino acid sequence BVH-P1 protein from S. pyogenes strain SPY57with a secretion signal at position 1 to 25; SEQ ID NO:6.

[0015]FIG. 7 is the DNA sequence of BVH-P1 gene from S. pyogenes strainB514 with a secretion signal at position 1 to 75; SEQ ID NO:7.

[0016]FIG. 8 is the amino acid sequence BVH-P1 protein from S. pyogenesstrain B514 with a secretion signal at position 1 to 25; SEQ ID NO:8.

[0017]FIG. 9 is the DNA sequence BVH-P1 gene without a secretion signalfrom serotype 3 S. pyogenes strain ATCC12384; SEQ ID NO:9.

[0018]FIG. 10 is the amino acid sequence BVH-P1 protein without asecretion signal from serotype 3 S. pyogenes strain ATCC12384 SEQ IDNO:10.

[0019]FIG. 11 is the DNA sequence BVH-P1 gene without a secretion signalfrom serotype 3 S. pyogenes strain LSPQ2699 (ATCC19615); SEQ ID NO:11.

[0020]FIG. 12 is the amino acid sequence BVH-P1 protein without asecretion signal from serotype 3 S. pyogenes strain LSPQ2699(ATCC19615); SEQ ID NO:12.

[0021]FIG. 13 is the DNA sequence BVH-P1 gene without a secretion signalfrom serotype 3 S. pyogenes strain SPY57; SEQ ID NO:13.

[0022]FIG. 14 is the amino acid sequence BVH-P1 protein without asecretion signal from serotype 3 S. pyogenes strain SPY57; SEQ ID NO:14.

[0023]FIG. 15 is the DNA sequence BVH-P1 gene without a secretion signalfrom serotype 3 S. pyogenes strain B514; SEQ ID NO:15.

[0024]FIG. 16 is the amino acid sequence BVH-P1 protein without asecretion signal from serotype 3 S. pyogenes strain B514; SEQ ID NO:16.

[0025] FIG. 17 depicts the comparison of the nucleotide sequences of theBVH-P1 genes from ATCC12384, LSPQ2699(ATCC19615), SPY57, B514, ATCC70029 (Oklahoma) and T28/51/4 (UO9352) S. pyogenes strains by using theprogram Clustal W from MacVector sequence analysis software (version6.5). Underneath the alignment, there is a consensus line. Shadednucleotides are identical between every sequences and gaps in thesequence introduced by alignment are indicated by hyphens.

[0026] FIG. 18 depicts the comparison of the predicted amino acidsequences of the BVH-P1 open reading frames from ATCC12384,LSPQ2699(ATCC19615), SPY57, B514, ATCC 70029 (Oklahoma) and T28/51/4(UO9352) S. pyogenes strains by using the program Clustal W fromMacVector sequence analysis software (version 6.5). Underneath thealignment, there is a consensus line. Shaded amino acid residues areidentical between every sequences and gaps in the sequence introduced byalignment are indicated by hyphens.

[0027]FIG. 19 is the DNA sequence of a gene from S. pneumonia; SEQ IDNO:17.

[0028]FIG. 20 is the amino acid sequence of a protein from S. pneumonia;SEQ ID NO:18.

DETAILED DESCRIPTION OF THE INVENTION

[0029] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 70%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analogues orderivatives thereof.

[0030] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 95%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analogues orderivatives thereof.

[0031] According to one aspect, the present invention relates topolypeptides characterized by the amino acid sequence comprising SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analogues orderivatives thereof.

[0032] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide capable of generatingantibodies having binding specificity for a polypeptide comprising asequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 orfragments, analogues or derivatives thereof.

[0033] In accordance with the present invention, there is provided aconsensus nucleotide sequence depicted in FIG. 17. As can be seen by thealignement, the polynucleotide encoding the polypeptide of the inventionis well conserved. Without restricting the scope of the invention, thefollowing table 1 shows the possible modifications. SEQ ID NO:19 coversthe consensus nucleotide sequence depicted in FIG. 17 with themodifications illustrated in Table 1: Position on alignement in Possiblenucleotide  21 C or T  53 C or T  69 G or A 103 G or C 149 C or T 150 Aor T 195 G or A 244 T or C 273 A or C 282 T or C 302 C or A 318 A or G334 G or T 394 C or T 400 G or A 415 C or T 428-448[CTGATGTCCCAACGACACCAT] or none 450 C or A 473 C or T 501 G or A 527 Tor C 572 T or A 573 T or A 595 A or C 596 C or G 597 G or C 630 A or G632 A or C 633 C or T 634 C or T 665 A or G 666 G or A 683 T or C 708 Cor T 733 [CAGATGTTAACT] or none 798 T or C 883 G or none 927 T or A 930T or C 943 T or none 952 T or A 955 G or A 964 T or C 973 G or A 976 Tor G 978 A or T 979 A or T 981 A or G 982 T or C 986 G or A 988 T or G1033  G or C 1034  C or G 1102  C or T 1143  A or T 1144  A or T 1145  Aor T 1146  A or T

[0034] In accordance with the present invention, there is provided aconsensus amino acid sequence depicted in FIG. 18. As can be seen by thealignement, the polypeptide of the invention is well conserved. Withoutrestricting the scope of the invention, the following table 2 shows thepossible modifications. SEQ ID NO:20 covers the consensus nucleotidesequence depicted in FIG. 18 with the modifications illustrated in Table2: Position on alignement in Possible amino acid  18 A or V  35 E or Q 50 T or I 101 T or N 112 A or S 132 P or S 134 V or I 139 S or P 143 to149 SDVPTTP or none 150 F or L 158 S or P 176 L or S 191 V or E 199 T orP or S 211 D or A 212 P or S 222 E or G 228 V or A 242 to 245 ETSQ ornone 246 E or M 247 T or L 248 S or T 295 A or L 296 S or L 297 A or P298 F or L 299 G or V 300 I or L 301 T or R 302 S or H 303 F or L 304 Sor V 305 G or V 306 Y or T 307 R or V 308 P or Q 309 G or E 310 D or I311 P or Q 312 G or E 313 D or I 314 H or I 326 E or V 327 N or S 329 Aor T 344 E or D 345 R or G 380 E or V 381 N or F

[0035] In accordance with the present invention, all polynucleotidesencoding polypeptides are within the scope of the present invention.

[0036] In a further embodiment, the polypeptides in accordance with thepresent invention are antigenic.

[0037] In a further embodiment, the polypeptides in accordance with thepresent invention are immunogenic.

[0038] In a further embodiment, the polypeptides in accordance with thepresent invention can elicit an immune response in an individual.

[0039] In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity to the polypeptides of the present invention as definedabove.

[0040] An antibody that “has binding specificity” is an antibody thatrecognizes and binds the selected polypeptide but which does notsubstantially recognize and bind other molecules in a sample, e.g., abiological sample. Specific binding can be measured using an ELISA assayin which the selected polypeptide is used as an antigen.

[0041] In accordance with the present invention, “protection” in thebiological studies is defined by a significant increase in the survivalcurve, rate or period. Statistical analysis using the Log rank test tocompare survival curves, and Fisher exact test to compare survival ratesand numbers of days to death, respectively, might be useful to calculateP values and determine whether the difference between the two groups isstatistically significant. P values of 0.05 are regarded as notsignificant.

[0042] As used herein, “fragments”, “analogues” or “derivatives” of thepolypeptides of the invention include those polypeptides in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably conserved) and which may benatural or unnatural. In one embodiment, derivatives and analogues ofpolypeptides of the invention will have about 70% identity with thosesequences illustrated in the figures or fragments thereof. That is, 70%of the residues are the same. In a further embodiment, polypeptides willhave greater than 75% homology. In a further embodiment, polypeptideswill have greater than 80% homology. In a further embodiment,polypeptides will have greater than 85% homology. In a furtherembodiment, polypeptides will have greater than 90% homology. In afurther embodiment, polypeptides will have greater than 95% homology. Ina further embodiment, polypeptides will have greater than 99% homology.In a further embodiment, derivatives and analogues of polypeptides ofthe invention will have less than about 20 amino acid residuesubstitutions, modifications or deletions and more preferably less than10. Preferred substitutions are those known in the art as conserved i.e.the substituted residues share physical or chemical properties such ashydrophobicity, size, charge or functional groups.

[0043] The skilled person will appreciate that fragments, analogues orderivatives of the proteins or polypeptides of the invention will alsofind use in the context of the present invention, i.e. asantigenic/immunogenic material. Thus, for instance proteins orpolypeptides which include one or more additions, deletions,substitutions or the like are encompassed by the present invention. Inaddition, it may be possible to replace one amino acid with another ofsimilar “type”. For instance replacing one hydrophobic amino acid withanother hydropholic amino acid.

[0044] One can use a program such as the CLUSTAL program to compareamino acid sequences. This program compares amino acid sequences andfinds the optimal alignment by inserting spaces in either sequence asappropriate. It is possible to calculate amino acid identity orsimilarity (identity plus conservation of amino acid type) for anoptimal alignment. A program like BLASTx will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of identity analysis arecontemplated in the present invention.

[0045] In an alternative approach, the analogues or derivatives could befusion proteins, incorporating moieties which render purificationeasier, for example by effectively tagging the desired protein orpolypeptide, it may be necessary to remove the “tag” or it may be thecase that the fusion protein itself retains sufficient antigenicity tobe useful.

[0046] In an additional aspect of the invention there are providedantigenic/immunogenic fragments of the proteins or polypeptides of theinvention, or of analogues or derivatives thereof.

[0047] The fragments of the present invention should include one or moreepitopic regions or be sufficiently similar to such regions to retaintheir antigenic/immunogenic properties. Thus, for fragments according tothe present invention the degree of identity is perhaps irrelevant,since they may be 100% identical to a particular part of a protein orpolypeptide, homologue or derivative as described herein. The key issue,once again, is that the fragment retains the antigenic/immunogenicproperties.

[0048] Thus, what is important for analogues, derivatives and fragmentsis that they possess at least a degree of the antigenicity/immunogenicof the protein or polypeptide from which they are derived.

[0049] Also included are polypeptides which have fused thereto othercompounds which alter the polypeptides biological or pharmacologicalproperties i.e. polyethylene glycol (PEG) to increase half-life; leaderor secretory amino acid sequences for ease of purification; prepro- andpro-sequences; and (poly)saccharides.

[0050] Furthermore, in those situations where amino acid regions arefound to be polymorphic, it may be desirable to vary one or moreparticular amino acids to more effectively mimic the different epitopesof the different streptococcus strains.

[0051] Moreover, the polypeptides of the present invention can bemodified by terminal —NH₂ acylation (eg. by acetylation, or thioglycolicacid amidation, terminal carbosy amidation, e.g. with ammonia ormethylamine) to provide stability, increased hydrophobicity for linkingor binding to a support or other molecule.

[0052] Also contemplated are hetero and homo polypeptide multimers ofthe polypeptide fragments, analogues and derivatives. These polymericforms include, for example, one or more polypeptides that have beencross-linked with cross-linkers such as avidin/biotin, gluteraldehyde ordimethylsuperimidate. Such polymeric forms also include polypeptidescontaining two or more tandem or inverted contiguous sequences, producedfrom multicistronic mRNAs generated by recombinant DNA technology.

[0053] Preferably, a fragment, analog or derivative of a polypeptide ofthe invention will comprise at least one antigenic region i.e. at leastone epitope.

[0054] In order to achieve the formation of antigenic polymers (i.e.synthetic multimers), polypeptides may be utilized having bishaloacetylgroups, nitroarylhalides, or the like, where the reagents being specificfor thio groups. Therefore, the link between two mercapto groups of thedifferent peptides may be a single bond or may be composed of a linkinggroup of at least two, typically at least four, and not more than 16,but usually not more than about 14 carbon atoms.

[0055] In a particular embodiment, polypeptide fragments, analogues andderivatives of the invention do not contain a methionine (Met) startingresidue. Preferably, polypeptides will not incorporate a leader orsecretory sequence (signal sequence). The signal portion of apolypeptide of the invention may be determined according to establishedmolecular biological techniques. In general, the polypeptide of interestmay be isolated from a streptococcal culture and subsequently sequencedto determine the initial residue of the mature protein and therefore thesequence of the mature polypeptide.

[0056] According to another aspect, there are provided vaccinecompositions comprising one or more streptococcal polypeptides of theinvention in admixture with a pharmaceutically acceptable carrierdiluent or adjuvant. Suitable adjuvants include oils i.e. Freund'scomplete or incomplete adjuvant; salts i.e. AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄)₂, silica, kaolin, carbon polynucleotides i.e. poly IC and poly AU.Preferred adjuvants include QuilA and Alhydrogel. Vaccines of theinvention may be administered parenterally by injection, rapid infusion,nasopharyngeal absorption, dermoabsorption, or bucal or oral.Pharmaceutically acceptable carriers also include tetanus toxoid.

[0057] The term vaccine is also meant to include antibodies. Inaccordance with the present invention, there is also provided the use ofone or more antibodies having binding specificity for the polypeptidesof the present invention for the treatment or prophylaxis ofstreptococcus infection and/or diseases and symptoms mediated bystreptococcus infection.

[0058] Vaccine compositions of the invention are used for the treatmentor prophylaxis of streptococcal infection and/or diseases and symptomsmediated by streptococcal infection As described in P. R. Murray (Ed, inchief),E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken.Manual of Clinical Microbiology, ASM Press, Washington, D.C. sixthedition, 1995, 1482p which are herein incorporated by reference. In oneembodiment, vaccine compositions of the present invention are used forthe prophylaxis or treatment of pharyngitis, erysipelas and impetigo,scarlet fever, and invasive diseases such as bacteremia and necrotizingfasciitis and also toxic shock. In one embodiment, vaccine compositionsof the invention are used for the prophylaxis or treatment ofstreptococcus infection and/or diseases and symptoms mediated bystreptococcus infection, in particular group A streptococcus (pyogenes),group B streptococcus (GBS or agalactiae), S. pneumoniae, dysgalactiae,uberis, nocardia as well as Staphylococcus aureus. In a furtherembodiment, the streptococcus infection is Streptococcus pyogenes.

[0059] In a particular embodiment, vaccines are administered to thoseindividuals at risk of streptococcus infection such as infants, elderlyand immunocompromised individuals.

[0060] As used in the present application, the term “individuals”include mammals. In a further embodiment, the mammal is human.

[0061] Vaccine compositions are preferably in unit dosage form of about0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01 to 10μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with an intervalof about 1 to 6 week intervals between immunizations.

[0062] Vaccine compositions are preferably in unit dosage form of about0.1 μg to 10 mg and more preferably 1 μg to 1 mg and most preferably 10to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervalsbetween immunizations.

[0063] According to another aspect, there are provided polynucleotidesencoding polypeptides characterized by the amino acid sequence chosenfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments, analoguesor derivatives thereof.

[0064] In one embodiment, polynucleotides are those illustrated in SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 19 which may include the open readingframes (ORF), encoding polypeptides of the invention.

[0065] It will be appreciated that the polynucleotide sequencesillustrated in the figures may be altered with degenerate codons yetstill encode the polypeptides of the invention. Accordingly the presentinvention further provides polynucleotides which hybridize to thepolynucleotide sequences herein above described (or the complementsequences thereof) having 50% identity between sequences. In oneembodiment, at least 70% identity between sequences. In one embodiment,at least 75% identity between sequences. In one embodiment, at least 80%identity between sequences. In one embodiment, at least 85% identitybetween sequences. In one embodiment, at least 90% identity betweensequences. In a further embodiment, polynucleotides are hybridizableunder stringent conditions i.e. having at least 95% identity. In afurther embodiment, more than 97% identity.

[0066] Suitable stringent conditions for hybridation can be readilydetermined by one of skilled in the art (see for example Sambrook etal., (1989) Molecular cloning: A Laboratory Manual, 2^(nd) ed, ColdSpring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999)Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., N.Y.).

[0067] In a further embodiment, the present invention providespolynucleotides that hybridise under stringent conditions to either

[0068] (a) a DNA sequence encoding a polypeptide or

[0069] (b) the complement of a DNA sequence encoding a polypeptide;

[0070] wherein said polypeptide comprising a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments or analogues thereof.

[0071] In a further embodiment, the present invention providespolynucleotides that hybridise under stringent conditions to either

[0072] (a) a DNA sequence encoding a polypeptide or

[0073] (b) the complement of a DNA sequence encoding a polypeptide;

[0074] wherein said polypeptide comprises at least 10 contiguous aminoacid residues from a polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments or analoguesthereof.

[0075] In a further embodiment, polynucleotides are those encodingpolypeptides of the invention illustrated in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 20.

[0076] In a further embodiment, polynucleotides are those illustrated inSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 19 encoding polypeptides of theinvention.

[0077] As will be readily appreciated by one skilled in the art,polynucleotides include both DNA and RNA.

[0078] The present invention also includes polynucleotides complementaryto the polynucleotides described in the present application.

[0079] In a further aspect, polynucleotides encoding polypeptides of theinvention, or fragments, analogues or derivatives thereof, may be usedin a DNA immunization method. That is, they can be incorporated into avector which is replicable and expressible upon injection therebyproducing the antigenic polypeptide in vivo. For example polynucleotidesmay be incorporated into a plasmid vector under the control of the CMVpromoter which is functional in eukaryotic cells. Preferably the vectoris injected intramuscularly.

[0080] According to another aspect, there is provided a process forproducing polypeptides of the invention by recombinant techniques byexpressing a polynucleotide encoding said polypeptide in a host cell andrecovering the expressed polypeptide product. Alternatively, thepolypeptides can be produced according to established synthetic chemicaltechniques i.e. solution phase or solid phase synthesis of oligopeptideswhich are ligated to produce the full polypeptide (block ligation).

[0081] General methods for obtention and evaluation of polynucleotidesand polypeptides are described in the following references: Sambrook etal, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor,N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols,from Molecular Cloning to Genetic Engineering, Edited by White B. A.,Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification,Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rdEdition, 1993, 380 pages; Current Protocols in Immunology, Edited byColigan J. E. et al., John Wiley & Sons Inc., New York which are hereinincorporated by reference.

[0082] For recombinant production, host cells are transfected withvectors which encode the polypeptide, and then cultured in a nutrientmedia modified as appropriate for activating promoters, selectingtransformants or amplifying the genes. Suitable vectors are those thatare viable and replicable in the chosen host and include chromosomal,non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA. The polypeptide sequence may beincorporated in the vector at the appropriate site using restrictionenzymes such that it is operably linked to an expression control regioncomprising a promoter, ribosome binding site (consensus region orShine-Dalgarno sequence), and optionally an operator (control element).One can select individual components of the expression control regionthat are appropriate for a given host and vector according toestablished molecular biology principles (Sambrook et al, MolecularCloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989;Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al.,John. Wiley and Sons, Inc. New York incorporated herein by reference).Suitable promoters include but are not limited to LTR or SV40 promoter,E. coli lac, tac or trp promoters and the phage lambda P_(L) promoter.Vectors will preferably incorporate an origin of replication as well asselection markers i.e. ampicilin resistance gene. Suitable bacterialvectors include pET, pQE70, pQE60, pQE-9, pbs, pD10 phagescript,psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a,pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII,pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL. Hostcells may be bacterial i.e. E. coli, Bacillus subtilis, Streptomyces;fungal i.e. Aspergillus niger, Aspergillus nidulins; yeast i.e.Saccharomyces or eukaryotic i.e. CHO, COS.

[0083] Upon expression of the polypeptide in culture, cells aretypically harvested by centrifugation then disrupted by physical orchemical means (if the expressed polypeptide is not secreted into themedia) and the resulting crude extract retained to isolate thepolypeptide of interest. Purification of the polypeptide from culturemedia or lysate may be achieved by established techniques depending onthe properties of the polypeptide i.e. using ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography and lectin chromatography. Finalpurification may be achieved using HPLC.

[0084] The polypeptide may be expressed with or without a leader orsecretion sequence. In the former case the leader may be removed usingpost-translational processing (see U.S. Pat. No. 4,431,739; U.S. Pat.No. 4,425,437; and U.S. Pat. No. 4,338,397 incorporated herein byreference) or be chemically removed subsequent to purifying theexpressed polypeptide.

[0085] According to a further aspect, the streptococcal polypeptides ofthe invention may be used in a diagnostic test for streptococcusinfection, in particular Streptococcus pyogenes infection. Severaldiagnostic methods are possible, for example detecting streptococcusorganism in a biological sample, the following procedure may befollowed:

[0086] a) obtaining a biological sample from an individual;

[0087] b) incubating an antibody or fragment thereof reactive with astreptococcus polypeptide of the invention with the biological sample toform a mixture; and

[0088] c) detecting specifically bound antibody or bound fragment in themixture which indicates the presence of streptococcus.

[0089] Alternatively, a method for the detection of antibody specific toa streptococcus antigen in a biological sample containing or suspectedof containing said antibody may be performed as follows:

[0090] a) obtaining a biological sample from an individual;

[0091] b) incubating one or more streptococcus polypeptides of theinvention or fragments thereof with the biological sample to form amixture; and

[0092] c) detecting specifically bound antigen or bound fragment in themixture which indicates the presence of antibody specific tostreptococcus.

[0093] One of skill in the art will recognize that this diagnostic testmay take several forms, including an immunological test such as anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latexagglutination assay, essentially to determine whether antibodiesspecific for the protein are present in an individual.

[0094] The DNA sequences encoding polypeptides of the invention may alsobe used to design DNA probes for use in detecting the presence ofstreptococcus in a biological sample suspected of containing suchbacteria. The detection method of this invention comprises:

[0095] a) obtaining the biological sample from an individual;

[0096] b) incubating one or more DNA probes having a DNA sequenceencoding a polypeptide of the invention or fragments thereof with thebiological sample to form a mixture; and

[0097] c) detecting specifically bound DNA probe in the mixture whichindicates the presence of streptococcus bacteria.

[0098] The DNA probes of this invention may also be used for detectingcirculating streptococcus i.e. Streptococcus pyogenes nucleic acids in asample, for example using a polymerase chain reaction, as a method ofdiagnosing streptococcus infections. The probe may be synthesized usingconventional techniques and may be immobilized on a solid phase, or maybe labelled with a detectable label. A preferred DNA probe for thisapplication is an oligomer having a sequence complementary to at leastabout 6 contiguous nucleotides of the Streptococcus pyogenespolypeptides of the invention.

[0099] Another diagnostic method for the detection of streptococcus inan individual comprises:

[0100] a) labelling an antibody reactive with a polypeptide of theinvention or fragment thereof with a detectable label;

[0101] b) administering the labelled antibody or labelled fragment tothe patient; and

[0102] c) detecting specifically bound labelled antibody or labelledfragment in the patient which indicates the presence of streptococcus.

[0103] A further aspect of the invention is the use of the streptococcuspolypeptides of the invention as immunogens for the production ofspecific antibodies for the diagnosis and in particular the treatment ofstreptococcus infection. Suitable antibodies may be determined usingappropriate screening methods, for example by measuring the ability of aparticular antibody to passively protect against streptococcus infectionin a test model. One example of an animal model is the mouse modeldescribed in the examples herein. The antibody may be a whole antibodyor an antigen-binding fragment thereof and may belong to anyimmunoglobulin class. The antibody or fragment may be of animal origin,specifically of mammalian origin and more specifically of murine, rat orhuman origin. It may be a natural antibody or a fragment thereof, or ifdesired, a recombinant antibody or antibody fragment. The termrecombinant antibody or antibody fragment means antibody or antibodyfragment which was produced using molecular biology techniques. Theantibody or antibody fragments may be polyclonal, or preferablymonoclonal. It may be specific for a number of epitopes associated withthe Streptococcus pyogenes polypeptides but is preferably specific forone.

[0104] A further aspect of the invention is the use of the antibodiesdirected to the streptococcus polypeptides of the invention for passiveimmunization. One could use the antibodies described in the presentapplication. Suitable antibodies may be determined using appropriatescreening methods, for example by measuring the ability of a particularantibody to passively protect against streptococcus infection in a testmodel. One example of an animal model is the mouse model described inthe examples herein. The antibody may be a whole antibody or anantigen-binding fragment thereof and may belong to any immunoglobulinclass. The antibody or fragment may be of animal origin, specifically ofmammalian origin and more specifically of murine, rat or human origin.It may be a natural antibody or a fragment thereof, or if desired, arecombinant antibody or antibody fragment. The term recombinant antibodyor antibody fragment means antibody or antibody fragment which wasproduced using molecular biology techniques. The antibody or antibodyfragments may be polyclonal, or preferably monoclonal. It may bespecific for a number of epitopes associated with the streptococcuspneumoniae polypeptides but is preferably specific for one.

[0105] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLE 1

[0106] This example illustrates the cloning of S. pyogenes gene.

[0107] The coding region of S. pyogenes gene BVH-P1 (SEQ ID NO:1) wasamplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA of serotype 3 S. pyogenesstrain ATCC12384 using the following oligos that contained baseextensions for the addition of restriction sites NcoI (CCATGG) and XhoI(CTCGAG): DMAR16 (5′-CAGGCCATGGAGTGGACACCACGATCGGTTAC-3′); DMAR17(5′-GCCGCTCGAGAGCATTAAAGGAGACATGAACATGATC-3′). PCR products werepurified from agarose gel using a QIAquick gel extraction kit fromQIAgen following the manufacturer's instructions (Chatsworth, Calif.),and digested with NcoI and XhoI (Pharmacia Canada Inc, Baie d'Urfé,Canada). The pET-21d(+) vector (Novagen, Madison, Wis.) was digestedwith NcoI and XhoI and purified from agarose gel using a QIAquick gelextraction kit from QIAgen (Chatsworth, Calif.). The NcoI-XhoI PCRproducts were ligated to the NcoI-XhoI pET-21d(+)expression vector. Theligated products were transformed into E. coli strain E. coli strainDH5α [φ80dlacZΔM15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17(r_(K)-m_(K)+)deoR thi-1 supE44 λ⁻gyrA96 relA1] (Gibco BRL, Gaithersburg, Md.)according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D. M.Glover (ed), pp. 109-135). Recombinant pET-21d(+)plasmid (rpET21d(+))containing BVH-P1 gene was purified using a QIAgen plasmid kit(Chatsworth, Calif.) and DNA insert was sequenced (Taq Dye DeoxyTerminator Cycle Sequencing kit, ABI, Foster City, Calif.).

[0108] It was determined that the open reading frame (ORF) which codesfor BVH-P1 contains 1170-bp and encodes a 389 amino acid residuespolypeptide with a predicted pI of 4.37 and a predicted molecular massof 41054 Da.

[0109] Analysis of the predicted amino acid residues sequence (SEQ IDNO:2)using the Spscan sofware (Wisconsin Sequence Analysis Package;Genetics Computer Group) suggested the existence of a 25 amino acidresidues signal peptide (MIITKKSLFVTSVALSLAPLATAQA), which ends with acleavage site situated between an alanine and a glutamine residues.Analysis of this ORF did not revealed the presence of repetitivestructures, cell wall anchoring motif (LPXTG), or IgA binding motif(MLKKIE).

[0110] An ORF which shares 62% with the S. pyogenes BVH-P1 gene wasinitially presented in the patent application PCT/CA99/00114 whichdescribed Group B streptococcus antigens. BVH-PL gene was also found toshare homology (62% identity) with an ORF present in the genome of S.pneumoniae (The Institute for Genomic Research).

EXAMPLE 2

[0111] This example describes the PCR amplification and sequencing ofBVH-P1 gene from other S. pyogenes strains and the evaluation of thelevel of molecular conservation of this gene.

[0112] Lancefield's serogroup A S. pyogenes LSPQ2296 (ATCC 19615) wasprovided by the laboratoire de la santé publique du Québec,Sainte-Anne-de-Bellevue; serotype 1 S. pyogenes SPY57 clinical isolatewas provided by the centre de recherche en infectiologie du centrehospitalier de l'universit{acute over (e )} Laval, Sainte-Foy; and S.pyogenes strain B514 which was initially isolated from a mouse wasprovided by Susan Hollingshead, from University of Alabama, Birmingham.The respective coding region of S. pyogenes gene BVH-P1 from strainsATCC 12384 (SEQ ID NO:1), LSPQ2699(ATCC19615) (SEQ ID NO:3), SPY57 (SEQID NO:5), and B514 (SEQ ID NO:7) were amplified by PCR (DNA ThermalCycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, Calif.) frombacterial cell lysates using the following oligos DMAR69(5′-CTGGGAAGATTATCTAGCACATTAATAC-3′); DMAR72(5′-CATAACGTTAAAACTGTCTAAAGGG-3′). PCR products were purified fromagarose gel using a QIAquick gel extraction kit from QIAgen followingthe manufacturer's instructions (Chatsworth, Calif.) and the DNA insertwere sequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI,Foster City, Calif.). The predicted amino acid sequences from strainsATCC12384 (SEQ ID NO:2), LSPQ2699(ATCC19615) (SEQ ID NO:4), SPY57 (SEQID NO:6), and B514 (SEQ ID NO:8) were respectively presented in thefollowing FIGS. 2, 4, 6, and 8.

[0113] The FIGS. 17 and 18 respectively depict the consensus nucleotideand predicted amino acid sequences established for S. pyogenes BVH-P1.In addition to the sequences presented herewith, the BVH-P1 genesequences from the genome sequencing project at the University ofOklahoma (serotype M1 S. pyogenes strain ATCC 70029:http://dnal.chem.ou.edu/strep.html) and from (Kil et al. 1994. Infect.Immun. 62:2440-2449: GenBank accession number U09352) were alsoincluded. No function or role in the pathogenesis of the bacteria orprotection against infection was described by Kil et al. for thesequence with GenBank accession number U09352. This latter sequencepresented by Kil et al. was shown to be located upstream of a S.pyogenes 67 kDa myosin-cross-reactive antigen.

[0114] Pairwise comparison of the BVH-P1 predicted protein sequencesrevealed between 95 to 100% identity with the exception of the BVH-P1sequence obtained from GenBank under the accesssion number U09352.Pairwise comparison of that particular sequence with the other fiveBVH-P1 sequences indicated identity between 87 to 91%. This lowerhomology can be explained by the presence of two regions (119-124 and262-281) which are more divergent comparatively to the other BVH-P1 genesequences. Beside these two regions in the BVH-P1 sequence obtained fromGenBank under the accesssion number U09352, the BVH-P1 genes showedgreat similarity in overall organization.

EXAMPLE 3

[0115] This example illustrates the cloning of S. pyogenes protein genein CMV plasmid pCMV-GH.

[0116] The DNA coding region of a S. pyogenes protein was inserted inphase downstream of a human growth hormone (hGH) gene which was underthe transcriptional control of the cytomegalovirus (CMV) promotor in theplasmid vector pCMV-GH (Tang et al., Nature, 1992, 356:152). The CMVpromotor is a non functional plasmid in E. coli cells but is active uponadministration of the plasmid in eukaryotic cells. The vector alsoincorporated the ampicillin resistance gene.

[0117] The coding region of BVH-P1 gene (SEQ ID NO:9) without its leaderpeptide region was amplified by PCR (DNA Thermal Cycler GeneAmp PCRsystem 2400 Perkin Elmer, San Jose, Calif.) from genomic DNA of serotype3 S. pyogenes strain ATCC12384 using the following oligos that containedbase extensions for the addition of restriction sites BamHI (GGATCC) andSalI (GTCGAC): DMAR24 (5′-TACCCGGATCCCCAAGAGTGGACACCACGATCGG-3′); DMAR25(5′-GCGCTCGTCGACGCGTATCTCAGCCTCTTATAGGGC-3′). The PCR product waspurified from agarose gel using a QIAquick gel extraction kit fromQIAgen (Chatsworth, Calif.), digested with restriction enzymes(Pharmacia Canada Inc, Baie d'Urfe, Canada). The pCMV-GH vector(Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, TheUniversity of Texas, Dallas, Tex.) was digested with BamHI and SalI andpurified from agarose gel using the QIAquick gel extraction kit fromQIAgen (Chatsworth, Calif.). The BamHI-SalI DNA fragments were ligatedto the BamHI-SalI pCMV-GH vector to create the hGH-BVH-P1 fusion proteinunder the control of the CMV promoter. The ligated products weretransformed into E. coli strain DH5α [φ80dlacZΔM15 Δ(lacZYA-argF)U169endA1 recA1 hsdR17(r_(K)-m_(K)+) deoR thi-1 supE44 λ⁻gyrA96 relA1](Gibco BRL, Gaithersburg, Md.) according to the method of Simanis(Hanahan, D. DNA Cloning, 1985, D. M. Glover (ed), pp. 109-135). Therecombinant pCMV plasmid was purified using a QIAgen plasmid kit(Chatsworth, Calif.) and the nucleotide sequence of the DNA insert wasverified by DNA sequencing.

EXAMPLE 4

[0118] This example illustrates the use of DNA to elicit an immuneresponse to S. pyogenes antigens.

[0119] A group of 8 female BALB/c mice (Charles River, St-Constant,Québec, Canada) were immunized by intramuscular injection of 100 μlthree times at two- or three-week intervals with 50 μg of recombinantPCMV-GH encoding BVH-P1 gene in presence of 50 μg ofgranulocyte-macrophage colony-stimulating factor (GM-CSF)-expressingplasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston,Department of Biochemistry, The University of Texas, Dallas, Tex.). Ascontrol, a group of mice were injected with 50 μg of pCMV-GH in presenceof 50 μg of pCMV-GH-GM-CSF. Blood samples were collected from theorbital sinus prior to each immunization and seven days following thethird injection and serum antibody responses were determined by ELISAusing purified BVH-P1-His•Tag from SEQ ID NO:11 S. pyogenes recombinantprotein as coating antigen.

EXAMPLE 5

[0120] This example illustrates the production and purification ofrecombinant S. pyogenes BVH-P1 protein.

[0121] The recombinant pET-21d(+)plasmid with BVH-P1 gene correspondingto the SEQ ID NO:9 was used to transform by electroporation (Gene PulserII apparatus, BIO-RAD Labs, Mississauga, Canada) E. coli strainBL21(DE3) (F⁻ompT hsdS_(B) (r⁻ _(B)m⁻ _(B)) gal dcm (DE3)) (Novagen,Madison, Wis.). In this strain of E. coli, the T7 promotor controllingexpression of the recombinant protein is specifically recognized by theT7 RNA polymerase (present on the λDE3 prophage) whose gene is under thecontrol of the lac promotor which is inducible byisopropyl-β-d-thio-galactopyranoside (IPTG). The transformantBL21(DE3)/rpET was grown at 37° C. with agitation at 250 rpm in LB broth(peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 100 μg ofcarbenicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml untilthe A₆₀₀ reached a value of 0.6. In order to induce the production of S.pyogenes BVH-P1-His•Tag recombinant protein (from SEQ ID NO:10), thecells were incubated for 3 additional hours in the presence of IPTG at afinal concentration of 1 mM. Induced cells from a 500 ml culture werepelleted by centrifugation and frozen at −70° C.

[0122] The purification of the recombinant proteins from the solublecytoplasmic fraction of IPTG-induced BL21(DE3)/rpET21b(+) was done byaffinity chromatography based on the properties of the His•Tag sequence(6 consecutive histidine residues) to bind to divalent cations (Ni²⁺)immobilized on the His•Bind metal chelation resin. Briefly, the pelletedcells obtained from a 500 mL culture induced with IPTG was resuspendedin lysis buffer (20 mM Tris, 500 mM NaCl. 10 mM imidazole, pH 7.9)containing 1 mM PMSF, sonicated and centrifuged at 12,000×g for 20 minto remove debris. The supernatant was deposited on a Ni-NTA agarosecolumn (Qiagen, Mississauga, Ontario, Canada). The S. pyogenesBVH-P1-His•Tag recombinant protein (from SEQ ID NO:10) was eluted with250 mM imidazole-500 mM NaCl-20 mM Tris pH 7.9. The removal of the saltand imidazole from the sample was done by dialysis against PBS at 4° C.The quantities of recombinant protein obtained from the soluble fractionof E. coli was estimated by MicroBCA (Pierce, Rockford, Ill.).

EXAMPLE 6

[0123] This example illustrates the accessibility to antibodies of theBVH-P1 protein at the surface of S. pyogenes strain.

[0124] Bacteria were grown in Tood Hewitt (TH) broth (DifcoLaboratories, Detroit Mich.) with 0.5% Yeast extract (DifcoLaboratories) and 0.5% peptone extract (Merck, Darmstadt, Germany) at37° C. in a 8% CO₂ atmosphere to give an OD_(490nm) of 0.600 (˜10⁸CFU/ml). Dilutions of anti-BVH-P1 or control sera were then added andallowed to bind to the cells, which were incubated for 2 h at 4° C.Samples were washed 4 times in blocking buffer [phosphate-bufferedsaline (PBS) containing 2% bovine serum albumin (BSA)], and then 1 ml ofgoat fluorescein (FITC)-conjugated anti-mouse IgG+IgM diluted inblocking buffer was added. After an additional incubation of 60 min atroom temperature, samples were washed 4 times in blocking buffer andfixed with 0.25% formaldehyde in PBS buffer for 18-24 h at 4° C. Cellswere washed 2 times in PBS buffer and resuspended in 500 μl of PBSbuffer. Cells were kept in the dark at 4° C. until analyzed by flowcytometry (Epics® XL; Beckman Coulter, Inc.). Flow cytometric analysisrevealed that BVH-P1-specific antibodies efficiently recognized theircorresponding surface exposed epitopes on both the homologous(ATCC12384; serotype3) and the heterologous (SPY57; seotype 1) S.pyogenes strains tested. It was determined that more than 90% of the10,000 S. pyogenes cells analyzed were labeled with the antobodiespresent in the BVH-MC1 specific anti-sera. These observations clearlydemonstrate that the BVH-P1 protein is accessible at the surface whereit can be easily recognized by antibodies. Anti-S. pyogenes antibodieswere shown to play an important role in the protection against S.pyogenes infection.

EXAMPLE 7

[0125] This example illustrates the protection against fatal S. pyogenesinfection induced by passive immunization of mice with rabbithyper-immune sera.

[0126] New Zealand rabbits (Charles River laboratories, Montreal,Canada) were injected subcutaneously at multiple sites withapproximately 50 μg and 100 μg of BVH-P1-His•Tag protein (from SEQ IDNO:10) that was produced and purified as described in Example 5 andadsorbed to Alhydrogel adjuvant (Superfos Biosector a/s). Rabbits wereimmunized three times at three-week intervals with the BVH-P1-His•Tagprotein (from SEQ ID NO:10). Blood samples were collected three weeksafter the third injection. The antibodies present in the serum werepurified by precipitation using 40% saturated ammonium sulfate. Groupsof 10 female CD-1 mice (Charles River) were injected intravenously with500 μl of purified serum collected either from BVH-P1-His•Tag (from SEQID NO:10) immunized rabbits or rabbits immunized with an unrelatedcontrol recombinant protein. Eighteen hours later the mice werechallenged with approximately 2×10⁷ CFU of the type 3 S. pyogenes strainATCC12384. Samples of the S. pyogenes challenge inoculum were plated onblood agar plates to determine the CFU and to verify the challenge dose.Deaths were recorded for a period of 5 days.

EXAMPLE 8

[0127] This example illustrates the protection of mice against fatal S.pyogenes infection induced by immunization with BVH-P1 protein.

[0128] Groups of 8 female CD-1 mice (Charles River) were immunizedsubcutaneously three times at three-week intervals with 20 μg ofaffinity purified S. pyogenes BVH-P1-His•Tag recombinant protein (fromSEQ ID NO:10) in presence of 10 μg of QuilA adjuvant (CedarlaneLaboratories Ltd, Hornby, Canada) or, as control, with QuilA adjuvantalone in PBS. Blood samples were collected from the orbital sinus on day1, 22 and 43 prior to each immunization and seven days (day 50)following the third injection. Analysis by ELISA using purifiedrecombinant BVH-P1 protein (from SEQ ID NO:10) clearly indicated thatthis protein is highly immunogenic in animals. Indeed reciprocal ELISAtiters higher than 10⁶ were determined for the mice immunized with thisrecombinant protein. Two weeks later the mice were challenged withapproximately 2×10⁷ CFU of the type 3 S. pyogenes strain ATCC12384.Samples of the S. pyogenes challenge inoculum were plated on blood agarplates to determine the CFU and to verify the challenge dose. Deathswere recorded for a period of 5 days. Five out of the 8 (62%) miceimmunized with three injections of 20 μg of purified recombinant BVH-P1(from SEQ ID NO:10) and QuilA adjuvant survived the bacterial challengeto only 2/7 (28%)in the control group. TABLE 3 Immunization of CD-1 micewith purified recombinant BVH-P1 protein confers protection againstsubsequent challenge with S. pyogenes strain ATCC 12384 Survival of themice challenged with S. pyogenes strain ATCC 12384 (Day after challenge:number of survivors/total number of mice challenged)) Groups 1 2 3 4 520 μg of 8/8 8/8 7/8 6/8 5/8 BVH-P1- His•Tag Control 7/7 6/7 3/7 2/7 2/7

[0129]

1 29 1 1170 DNA S. pyogenes 1 atgattatta ctaaaaagag cttatttgtgacaagtgtcg ctttgtcgtt agcacctttg 60 gcgacagcac aggcacaaga gtggacaccacgatcggtta cagaaatcaa gtctgaactc 120 gtcctagttg ataatgtttt tacttatactgtaaaatacg gtgacacttt aagcacaatt 180 gctgaagcaa tgggaattga tgtgcatgtcttaggagata ttaatcatat tgctaatatt 240 gacttaattt ttccagacac gatcctaacagccaactaca accaacacgg tcaggcaacg 300 actttgacgg ttcaagcgcc tgcttctagtccagctagcg ttagtcatgt acctagcagt 360 gagccattac cccaagcatc tgccacctctcaatcgactg ttcctatggc accatctgcg 420 acaccatctg atgtcccaac gacaccattcgcatctgcaa agccagatag ttctgtgaca 480 gcgtcatctg agctcacatc gtcaacgaatgatgtttcga ctgagttgtc tagcgaatca 540 caaaagcagc cagaagtacc acaagaagcagttccaactc ctaaagcagc tgaaacgact 600 gaagtcgaac ctaagacaga catctcagaggattcaactt cagctaatag gcctgtacct 660 aacgagagtg cttcagaaga agtttcttctgcggccccag cacaagcccc agcagaaaaa 720 gaagaaacct ctgcgccagc agcacaaaaagctgtagctg acaccacaag tgttgcaacc 780 tcaaatggcc tttcttacgc tccaaaccatgcctacaatc caatgaatgc agggcttcaa 840 ccacaaacag cagccttcaa agaagaagtggcttctgcct ttggtattac gtcatttagt 900 ggttaccgtc caggtgatcc aggagatcatggtaaaggtt tggccattga ttttatggtg 960 cctgaaaatt ctgctcttgg tgatcaagttgctcaatatg ccattgacca tatggcagag 1020 cgtggtattt catacgttat ttggaaacagcgattctatg cgccatttgc aagtatttac 1080 ggaccagcct acacatggaa ccccatgccagatcgcggca gtattacaga aaaccattat 1140 gatcatgttc atgtctcctt taatgcttaa1170 2 389 PRT S. pyogenes 2 Met Ile Ile Thr Lys Lys Ser Leu Phe Val ThrSer Val Ala Leu Ser 1 5 10 15 Leu Ala Pro Leu Ala Thr Ala Gln Ala GlnGlu Trp Thr Pro Arg Ser 20 25 30 Val Thr Glu Ile Lys Ser Glu Leu Val LeuVal Asp Asn Val Phe Thr 35 40 45 Tyr Thr Val Lys Tyr Gly Asp Thr Leu SerThr Ile Ala Glu Ala Met 50 55 60 Gly Ile Asp Val His Val Leu Gly Asp IleAsn His Ile Ala Asn Ile 65 70 75 80 Asp Leu Ile Phe Pro Asp Thr Ile LeuThr Ala Asn Tyr Asn Gln His 85 90 95 Gly Gln Ala Thr Thr Leu Thr Val GlnAla Pro Ala Ser Ser Pro Ala 100 105 110 Ser Val Ser His Val Pro Ser SerGlu Pro Leu Pro Gln Ala Ser Ala 115 120 125 Thr Ser Gln Ser Thr Val ProMet Ala Pro Ser Ala Thr Pro Ser Asp 130 135 140 Val Pro Thr Thr Pro PheAla Ser Ala Lys Pro Asp Ser Ser Val Thr 145 150 155 160 Ala Ser Ser GluLeu Thr Ser Ser Thr Asn Asp Val Ser Thr Glu Leu 165 170 175 Ser Ser GluSer Gln Lys Gln Pro Glu Val Pro Gln Glu Ala Val Pro 180 185 190 Thr ProLys Ala Ala Glu Thr Thr Glu Val Glu Pro Lys Thr Asp Ile 195 200 205 SerGlu Asp Ser Thr Ser Ala Asn Arg Pro Val Pro Asn Glu Ser Ala 210 215 220Ser Glu Glu Val Ser Ser Ala Ala Pro Ala Gln Ala Pro Ala Glu Lys 225 230235 240 Glu Glu Thr Ser Ala Pro Ala Ala Gln Lys Ala Val Ala Asp Thr Thr245 250 255 Ser Val Ala Thr Ser Asn Gly Leu Ser Tyr Ala Pro Asn His AlaTyr 260 265 270 Asn Pro Met Asn Ala Gly Leu Gln Pro Gln Thr Ala Ala PheLys Glu 275 280 285 Glu Val Ala Ser Ala Phe Gly Ile Thr Ser Phe Ser GlyTyr Arg Pro 290 295 300 Gly Asp Pro Gly Asp His Gly Lys Gly Leu Ala IleAsp Phe Met Val 305 310 315 320 Pro Glu Asn Ser Ala Leu Gly Asp Gln ValAla Gln Tyr Ala Ile Asp 325 330 335 His Met Ala Glu Arg Gly Ile Ser TyrVal Ile Trp Lys Gln Arg Phe 340 345 350 Tyr Ala Pro Phe Ala Ser Ile TyrGly Pro Ala Tyr Thr Trp Asn Pro 355 360 365 Met Pro Asp Arg Gly Ser IleThr Glu Asn His Tyr Asp His Val His 370 375 380 Val Ser Phe Asn Ala 3853 1182 DNA S. pyogenes 3 atgattatta ctaaaaagag cttatttgtg acaagtgtcgctttgtcgtt agcacctttg 60 gcgacagcgc aggcacaaga gtggacacca cgatcggttacagaaatcaa gtctgaactc 120 gtcctagttg ataatgtttt tacttatata gtaaaatacggtgacacttt aagcacaatt 180 gctgaagcaa tggggattga tgtgcatgtc ttaggagatattaatcatat tgctaatatt 240 gacttaattt ttccagacac gatcctaaca gcaaactacaaccaacacgg tcaggcaacg 300 actttgacgg ttcaagcacc tgcttctagt ccatctagcgttagtcatgt acctagcagt 360 gagccattac cccaagcatc tgccacctct caaccgactgttcctatggc accatctgcg 420 acaccatctg atgtcccaac gacaccattc gcatctgcaaagccagatag ttctgtgaca 480 gcgtcatctg agctcacatc gtcaacgaat gatgtttcgactgagttgtc tagcgaatca 540 caaaagcagc cagaagtacc acaagaagca gttccaactcctaaagcagc tgaaccgact 600 gaagtcgaac ctaagacaga catctcagaa gacccaacttcagctaatag gcctgtacct 660 aacgagagtg cttcagaaga agcttcttct gcggccccagcacaagctcc agcagaaaaa 720 gaagaaacct ctcagatgtt aactgcgcca gcagcacaaaaagctgtagc tgacaccaca 780 agtgttgcaa cctcaaacgg cctttcttac gctccaaaccatgcctacaa tccaatgaat 840 gcagggcttc aaccacaaac agcagccttc aaagaagaagtggcttctgc ctttggtatt 900 acgtcattta gtggttaccg tccaggagat ccaggagatcatggtaaagg attagccatt 960 gactttatgg taccggttag ctctacgctt ggtgatcaagttgctcaata tgccattgac 020 catatggcag agcgtggtat ttcatacgtt atttggaaacagcgattcta tgcgccattt 080 gcaagtattt acggaccagc ctacacatgg aaccccatgccagatcgcgg cagtattaca 140 gaaaaccatt atgatcatgt tcatgtctcc tttaatgctt aa182 4 393 PRT S. pyogenes 4 Met Ile Ile Thr Lys Lys Ser Leu Phe Val ThrSer Val Ala Leu Ser 1 5 10 15 Leu Ala Pro Leu Ala Thr Ala Gln Ala GlnGlu Trp Thr Pro Arg Ser 20 25 30 Val Thr Glu Ile Lys Ser Glu Leu Val LeuVal Asp Asn Val Phe Thr 35 40 45 Tyr Ile Val Lys Tyr Gly Asp Thr Leu SerThr Ile Ala Glu Ala Met 50 55 60 Gly Ile Asp Val His Val Leu Gly Asp IleAsn His Ile Ala Asn Ile 65 70 75 80 Asp Leu Ile Phe Pro Asp Thr Ile LeuThr Ala Asn Tyr Asn Gln His 85 90 95 Gly Gln Ala Thr Thr Leu Thr Val GlnAla Pro Ala Ser Ser Pro Ser 100 105 110 Ser Val Ser His Val Pro Ser SerGlu Pro Leu Pro Gln Ala Ser Ala 115 120 125 Thr Ser Gln Pro Thr Val ProMet Ala Pro Ser Ala Thr Pro Ser Asp 130 135 140 Val Pro Thr Thr Pro PheAla Ser Ala Lys Pro Asp Ser Ser Val Thr 145 150 155 160 Ala Ser Ser GluLeu Thr Ser Ser Thr Asn Asp Val Ser Thr Glu Leu 165 170 175 Ser Ser GluSer Gln Lys Gln Pro Glu Val Pro Gln Glu Ala Val Pro 180 185 190 Thr ProLys Ala Ala Glu Pro Thr Glu Val Glu Pro Lys Thr Asp Ile 195 200 205 SerGlu Asp Pro Thr Ser Ala Asn Arg Pro Val Pro Asn Glu Ser Ala 210 215 220Ser Glu Glu Ala Ser Ser Ala Ala Pro Ala Gln Ala Pro Ala Glu Lys 225 230235 240 Glu Glu Thr Ser Gln Met Leu Thr Ala Pro Ala Ala Gln Lys Ala Val245 250 255 Ala Asp Thr Thr Ser Val Ala Thr Ser Asn Gly Leu Ser Tyr AlaPro 260 265 270 Asn His Ala Tyr Asn Pro Met Asn Ala Gly Leu Gln Pro GlnThr Ala 275 280 285 Ala Phe Lys Glu Glu Val Ala Ser Ala Phe Gly Ile ThrSer Phe Ser 290 295 300 Gly Tyr Arg Pro Gly Asp Pro Gly Asp His Gly LysGly Leu Ala Ile 305 310 315 320 Asp Phe Met Val Pro Val Ser Ser Thr LeuGly Asp Gln Val Ala Gln 325 330 335 Tyr Ala Ile Asp His Met Ala Glu ArgGly Ile Ser Tyr Val Ile Trp 340 345 350 Lys Gln Arg Phe Tyr Ala Pro PheAla Ser Ile Tyr Gly Pro Ala Tyr 355 360 365 Thr Trp Asn Pro Met Pro AspArg Gly Ser Ile Thr Glu Asn His Tyr 370 375 380 Asp His Val His Val SerPhe Asn Ala 385 390 5 1170 DNA S. pyogenes 5 atgattatta ctaaaaagagcttatttgtg acaagtgtcg ctttgtcgtt agtacctttg 60 gcgacagcgc aggcacaagagtggacacca cgatcggtta cagaaatcaa gtctgaactc 120 gtcctagttg ataatgtttttacttatact gtaaaatacg gtgacacttt aagcacaatt 180 gctgaagcaa tggggattgatgtgcatgtc ttaggagata ttaatcatat tgctaatatt 240 gacctaattt ttccagacacgatcctaaca gcaaactaca atcaacacgg tcaggcaacg 300 aatttgacgg ttcaagcacctgcttctagt ccagctagcg ttagtcatgt acctagcagt 360 gagccattac cccaagcatctgccacctct caaccgactg ttcctatggc accacctgcg 420 acaccatctg atgtcccaacgacaccattc gcatctgcaa agccagatag ttctgtgaca 480 gcgtcatctg agctcacatcgtcaacgaat gatgtttcga ctgagttgtc tagcgaatca 540 caaaagcagc cagaagtaccacaagaagca gttccaactc ctaaagcagc tgaaacgact 600 gaagtcgaac ctaagacagacatctcagaa gccccaactt cagctaatag gcctgtacct 660 aacgagagtg cttcagaagaagtttcttct gcggccccag cacaagcccc agcagaaaaa 720 gaagaaacct ctgcgccagcagcacaaaaa gctgtagctg acaccacaag tgttgcaacc 780 tcaaatggcc tttcttacgctccaaaccat gcctacaatc caatgaatgc agggcttcaa 840 ccacaaacag cagccttcaaagaagaagtg gcttctgcct ttggtattac gtcatttagt 900 ggttaccgtc caggtgatccaggagatcat ggtaaaggtt tggccattga ttttatggtg 960 cgtggtattt catacgttatttggaaacag cgattctatg cgccatttgc aagtatttac 1080 ggaccagcct acacatggaaccccatgcca gatcgcggca gtattacaga aaaccattat 1140 gatcatgttc atgtctcctttaatgcttaa 1170 6 389 PRT S. pyogenes 6 Met Ile Ile Thr Lys Lys Ser LeuPhe Val Thr Ser Val Ala Leu Ser 1 5 10 15 Leu Val Pro Leu Ala Thr AlaGln Ala Gln Glu Trp Thr Pro Arg Ser 20 25 30 Val Thr Glu Ile Lys Ser GluLeu Val Leu Val Asp Asn Val Phe Thr 35 40 45 Tyr Thr Val Lys Tyr Gly AspThr Leu Ser Thr Ile Ala Glu Ala Met 50 55 60 Gly Ile Asp Val His Val LeuGly Asp Ile Asn His Ile Ala Asn Ile 65 70 75 80 Asp Leu Ile Phe Pro AspThr Ile Leu Thr Ala Asn Tyr Asn Gln His 85 90 95 Gly Gln Ala Thr Asn LeuThr Val Gln Ala Pro Ala Ser Ser Pro Ala 100 105 110 Ser Val Ser His ValPro Ser Ser Glu Pro Leu Pro Gln Ala Ser Ala 115 120 125 Thr Ser Gln ProThr Val Pro Met Ala Pro Pro Ala Thr Pro Ser Asp 130 135 140 Val Pro ThrThr Pro Phe Ala Ser Ala Lys Pro Asp Ser Ser Val Thr 145 150 155 160 AlaSer Ser Glu Leu Thr Ser Ser Thr Asn Asp Val Ser Thr Glu Leu 165 170 175Ser Ser Glu Ser Gln Lys Gln Pro Glu Val Pro Gln Glu Ala Val Pro 180 185190 Thr Pro Lys Ala Ala Glu Thr Thr Glu Val Glu Pro Lys Thr Asp Ile 195200 205 Ser Glu Ala Pro Thr Ser Ala Asn Arg Pro Val Pro Asn Glu Ser Ala210 215 220 Ser Glu Glu Val Ser Ser Ala Ala Pro Ala Gln Ala Pro Ala GluLys 225 230 235 240 Glu Glu Thr Ser Ala Pro Ala Ala Gln Lys Ala Val AlaAsp Thr Thr 245 250 255 Ser Val Ala Thr Ser Asn Gly Leu Ser Tyr Ala ProAsn His Ala Tyr 260 265 270 Asn Pro Met Asn Ala Gly Leu Gln Pro Gln ThrAla Ala Phe Lys Glu 275 280 285 Glu Val Ala Ser Ala Phe Gly Ile Thr SerPhe Ser Gly Tyr Arg Pro 290 295 300 Gly Asp Pro Gly Asp His Gly Lys GlyLeu Ala Ile Asp Phe Met Val 305 310 315 320 Pro Glu Asn Ser Ala Leu GlyAsp Gln Val Ala Gln Tyr Ala Ile Asp 325 330 335 His Met Ala Glu Arg GlyIle Ser Tyr Val Ile Trp Lys Gln Arg Phe 340 345 350 Tyr Ala Pro Phe AlaSer Ile Tyr Gly Pro Ala Tyr Thr Trp Asn Pro 355 360 365 Met Pro Asp ArgGly Ser Ile Thr Glu Asn His Tyr Asp His Val His 370 375 380 Val Ser PheAsn Ala 385 7 1149 DNA S. pyogenes 7 atgattatta ctaaaaagag cttatttgtgacaagtgtcg ctttgtcgtt agcacctttg 60 gcgacagcgc aggcacaaga gtggacaccacgatcggtta cagaaatcaa gtctgaactc 120 gtcctagttg ataatgtttt tacttatacagtaaaatacg gtgacacttt aagcacaatt 180 gctgaagcaa tggggattga tgtgcatgtcttaggagata ttaatcatat tgctaatatt 240 gacttaattt ttccagacac gatcctaacagcaaactaca atcaacacgg tcaggcaacg 300 actttgacgg ttcaagcacc tgcttctagtccagctagcg ttagtcatgt acctagcagt 360 gagccattac cccaagcatc tgccacctctcaaccgactg ttcctatggc accatctgcg 420 acaccattag catctgcaaa gccagatagttctgtgacag cgtcatctga gctcacatcg 480 tcaacgaatg atgtttcgac tgagtcgtctagcgaatcac aaaagcagcc agaagtacca 540 caagaagcag ttccaactcc taaagcagctgaaacgactg aagtcgaacc taagacagac 600 atctcagaag acccaacttc agctaataggcctgtaccta acgagagtgc ttcagaagaa 660 gtttcttctg cggccccagc acaagccccagcagaaaaag aagaaacctc tgcgccagca 720 gcacaaaaag ctgtagctga caccacaagtgttgcaacct caaacggcct ttcttacgct 780 ccaaaccatg cctacaatcc aatgaatgcagggcttcaac cacaaacagc agccttcaaa 840 gaagaagtgg cttctgcctt tggtattacgtcatttagtg gttaccgtcc aggtgaccca 900 ggagatcatg gtaaaggttt ggccattgattttatggtgc ctgaaaattc tgctcttggt 960 gatcaagttg ctcaatatgc cattgaccatatggcagagc gtggtatttc atacgttatt 1020 tggaaacagc gattctatgc gccatttgcaagtatttacg gaccagctta cacatggaac 1080 cccatgccag atcgcggcag tattacagaaaaccattatg atcatgttca tgtctccttt 1140 aatgcttaa 1149 8 382 PRT S.pyogenes 8 Met Ile Ile Thr Lys Lys Ser Leu Phe Val Thr Ser Val Ala LeuSer 1 5 10 15 Leu Ala Pro Leu Ala Thr Ala Gln Ala Gln Glu Trp Thr ProArg Ser 20 25 30 Val Thr Glu Ile Lys Ser Glu Leu Val Leu Val Asp Asn ValPhe Thr 35 40 45 Tyr Thr Val Lys Tyr Gly Asp Thr Leu Ser Thr Ile Ala GluAla Met 50 55 60 Gly Ile Asp Val His Val Leu Gly Asp Ile Asn His Ile AlaAsn Ile 65 70 75 80 Asp Leu Ile Phe Pro Asp Thr Ile Leu Thr Ala Asn TyrAsn Gln His 85 90 95 Gly Gln Ala Thr Thr Leu Thr Val Gln Ala Pro Ala SerSer Pro Ala 100 105 110 Ser Val Ser His Val Pro Ser Ser Glu Pro Leu ProGln Ala Ser Ala 115 120 125 Thr Ser Gln Pro Thr Val Pro Met Ala Pro SerAla Thr Pro Leu Ala 130 135 140 Ser Ala Lys Pro Asp Ser Ser Val Thr AlaSer Ser Glu Leu Thr Ser 145 150 155 160 Ser Thr Asn Asp Val Ser Thr GluSer Ser Ser Glu Ser Gln Lys Gln 165 170 175 Pro Glu Val Pro Gln Glu AlaVal Pro Thr Pro Lys Ala Ala Glu Thr 180 185 190 Thr Glu Val Glu Pro LysThr Asp Ile Ser Glu Asp Pro Thr Ser Ala 195 200 205 Asn Arg Pro Val ProAsn Glu Ser Ala Ser Glu Glu Val Ser Ser Ala 210 215 220 Ala Pro Ala GlnAla Pro Ala Glu Lys Glu Glu Thr Ser Ala Pro Ala 225 230 235 240 Ala GlnLys Ala Val Ala Asp Thr Thr Ser Val Ala Thr Ser Asn Gly 245 250 255 LeuSer Tyr Ala Pro Asn His Ala Tyr Asn Pro Met Asn Ala Gly Leu 260 265 270Gln Pro Gln Thr Ala Ala Phe Lys Glu Glu Val Ala Ser Ala Phe Gly 275 280285 Ile Thr Ser Phe Ser Gly Tyr Arg Pro Gly Asp Pro Gly Asp His Gly 290295 300 Lys Gly Leu Ala Ile Asp Phe Met Val Pro Glu Asn Ser Ala Leu Gly305 310 315 320 Asp Gln Val Ala Gln Tyr Ala Ile Asp His Met Ala Glu ArgGly Ile 325 330 335 Ser Tyr Val Ile Trp Lys Gln Arg Phe Tyr Ala Pro PheAla Ser Ile 340 345 350 Tyr Gly Pro Ala Tyr Thr Trp Asn Pro Met Pro AspArg Gly Ser Ile 355 360 365 Thr Glu Asn His Tyr Asp His Val His Val SerPhe Asn Ala 370 375 380 9 1095 DNA S. pyogenes 9 caagagtgga caccacgatcggttacagaa atcaagtctg aactcgtcct agttgataat 60 gtttttactt atactgtaaaatacggtgac actttaagca caattgctga agcaatggga 120 attgatgtgc atgtcttaggagatattaat catattgcta atattgactt aatttttcca 180 gacacgatcc taacagccaactacaaccaa cacggtcagg caacgacttt gacggttcaa 240 gcgcctgctt ctagtccagctagcgttagt catgtaccta gcagtgagcc attaccccaa 300 gcatctgcca cctctcaatcgactgttcct atggcaccat ctgcgacacc atctgatgtc 360 ccaacgacac cattcgcatctgcaaagcca gatagttctg tgacagcgtc atctgagctc 420 acatcgtcaa cgaatgatgtttcgactgag ttgtctagcg aatcacaaaa gcagccagaa 480 gtaccacaag aagcagttccaactcctaaa gcagctgaaa cgactgaagt cgaacctaag 540 acagacatct cagaggattcaacttcagct aataggcctg tacctaacga gagtgcttca 600 gaagaagttt cttctgcggccccagcacaa gccccagcag aaaaagaaga aacctctgcg 660 ccagcagcac aaaaagctgtagctgacacc acaagtgttg caacctcaaa tggcctttct 720 tacgctccaa accatgcctacaatccaatg aatgcagggc ttcaaccaca aacagcagcc 780 ttcaaagaag aagtggcttctgcctttggt attacgtcat ttagtggtta ccgtccaggt 840 gatccaggag atcatggtaaaggtttggcc attgatttta tggtgcctga aaattctgct 900 cttggtgatc aagttgctcaatatgccatt gaccatatgg cagagcgtgg tatttcatac 960 gttatttgga aacagcgattctatgcgcca tttgcaagta tttacggacc agcctacaca 1020 tggaacccca tgccagatcgcggcagtatt acagaaaacc attatgatca tgttcatgtc 1080 tcctttaatg cttaa 109510 364 PRT S. pyogenes 10 Gln Glu Trp Thr Pro Arg Ser Val Thr Glu IleLys Ser Glu Leu Val 1 5 10 15 Leu Val Asp Asn Val Phe Thr Tyr Thr ValLys Tyr Gly Asp Thr Leu 20 25 30 Ser Thr Ile Ala Glu Ala Met Gly Ile AspVal His Val Leu Gly Asp 35 40 45 Ile Asn His Ile Ala Asn Ile Asp Leu IlePhe Pro Asp Thr Ile Leu 50 55 60 Thr Ala Asn Tyr Asn Gln His Gly Gln AlaThr Thr Leu Thr Val Gln 65 70 75 80 Ala Pro Ala Ser Ser Pro Ala Ser ValSer His Val Pro Ser Ser Glu 85 90 95 Pro Leu Pro Gln Ala Ser Ala Thr SerGln Ser Thr Val Pro Met Ala 100 105 110 Pro Ser Ala Thr Pro Ser Asp ValPro Thr Thr Pro Phe Ala Ser Ala 115 120 125 Lys Pro Asp Ser Ser Val ThrAla Ser Ser Glu Leu Thr Ser Ser Thr 130 135 140 Asn Asp Val Ser Thr GluLeu Ser Ser Glu Ser Gln Lys Gln Pro Glu 145 150 155 160 Val Pro Gln GluAla Val Pro Thr Pro Lys Ala Ala Glu Thr Thr Glu 165 170 175 Val Glu ProLys Thr Asp Ile Ser Glu Asp Ser Thr Ser Ala Asn Arg 180 185 190 Pro ValPro Asn Glu Ser Ala Ser Glu Glu Val Ser Ser Ala Ala Pro 195 200 205 AlaGln Ala Pro Ala Glu Lys Glu Glu Thr Ser Ala Pro Ala Ala Gln 210 215 220Lys Ala Val Ala Asp Thr Thr Ser Val Ala Thr Ser Asn Gly Leu Ser 225 230235 240 Tyr Ala Pro Asn His Ala Tyr Asn Pro Met Asn Ala Gly Leu Gln Pro245 250 255 Gln Thr Ala Ala Phe Lys Glu Glu Val Ala Ser Ala Phe Gly IleThr 260 265 270 Ser Phe Ser Gly Tyr Arg Pro Gly Asp Pro Gly Asp His GlyLys Gly 275 280 285 Leu Ala Ile Asp Phe Met Val Pro Glu Asn Ser Ala LeuGly Asp Gln 290 295 300 Val Ala Gln Tyr Ala Ile Asp His Met Ala Glu ArgGly Ile Ser Tyr 305 310 315 320 Val Ile Trp Lys Gln Arg Phe Tyr Ala ProPhe Ala Ser Ile Tyr Gly 325 330 335 Pro Ala Tyr Thr Trp Asn Pro Met ProAsp Arg Gly Ser Ile Thr Glu 340 345 350 Asn His Tyr Asp His Val His ValSer Phe Asn Ala 355 360 11 1106 DNA S. pyogenes 11 caagagtgga caccacgatcggttacagaa atcaagtctg aactcgtcct agttgataat 60 gtttttactt atatagtaaaatacggtgac actttaagca caattgctga agcaatgggg 120 attgatgtgc atgtcttaggagatattaat catattgcta atattgactt aatttttcca 180 gacacgatcc taacagcaaactacaaccaa cacggtcagg caacgacttt gacggttcaa 240 gcacctgctt ctagtccatctagcgttagt catgtaccta gcagtgagcc attaccccaa 300 gcatctgcca cctctcaaccgactgttcct atggcaccat ctgcgacacc atctgatgtc 360 ccaacgacac cattcgcatctgcaaagcca gatagttctg tgacagcgtc atctgagctc 420 acatcgtcaa cgaatgatgtttcgactgag ttgtctagcg aatcacaaaa gcagccagaa 480 gtaccacaag aagcagttccaactcctaaa gcagctgaac cgactgaagt cgaacctaag 540 acagacatct cagaagacccaacttcagct aataggcctg acctaacgag agtgcttcag 600 aagaagcttc ttctgcggccccagcacaag ctccagcaga aaaagaagaa acctctcaga 660 tgttaactgc gccagcagcacaaaaagctg tagctgacac cacaagtgtt gcaacctcaa 720 acggcctttc ttacgctccaaaccatgcct acaatccaat gaatgcaggg cttcaaccac 780 aaacagcagc cttcaaagaagaagtggctt ctgcctttgg tattacgtca tttagtggtt 840 accgtccagg agatccaggagatcatggta aaggattagc cattgacttt atggtaccgg 900 ttagctctac gcttggtgatcaagttgctc aatatgccat tgaccatatg gcagagcgtg 960 gtatttcata cgttatttggaaacagcgat tctatgcgcc atttgcaagt atttacggac 1020 cagcctacac atggaaccccatgccagatc gcggcagtat tacagaaaac cattatgatc 1080 atgttcatgt ctcctttaatgcttaa 1106 12 368 PRT S. pyogenes 12 Gln Glu Trp Thr Pro Arg Ser ValThr Glu Ile Lys Ser Glu Leu Val 1 5 10 15 Leu Val Asp Asn Val Phe ThrTyr Ile Val Lys Tyr Gly Asp Thr Leu 20 25 30 Ser Thr Ile Ala Glu Ala MetGly Ile Asp Val His Val Leu Gly Asp 35 40 45 Ile Asn His Ile Ala Asn IleAsp Leu Ile Phe Pro Asp Thr Ile Leu 50 55 60 Thr Ala Asn Tyr Asn Gln HisGly Gln Ala Thr Thr Leu Thr Val Gln 65 70 75 80 Ala Pro Ala Ser Ser ProSer Ser Val Ser His Val Pro Ser Ser Glu 85 90 95 Pro Leu Pro Gln Ala SerAla Thr Ser Gln Pro Thr Val Pro Met Ala 100 105 110 Pro Ser Ala Thr ProSer Asp Val Pro Thr Thr Pro Phe Ala Ser Ala 115 120 125 Lys Pro Asp SerSer Val Thr Ala Ser Ser Glu Leu Thr Ser Ser Thr 130 135 140 Asn Asp ValSer Thr Glu Leu Ser Ser Glu Ser Gln Lys Gln Pro Glu 145 150 155 160 ValPro Gln Glu Ala Val Pro Thr Pro Lys Ala Ala Glu Pro Thr Glu 165 170 175Val Glu Pro Lys Thr Asp Ile Ser Glu Asp Pro Thr Ser Ala Asn Arg 180 185190 Pro Val Pro Asn Glu Ser Ala Ser Glu Glu Ala Ser Ser Ala Ala Pro 195200 205 Ala Gln Ala Pro Ala Glu Lys Glu Glu Thr Ser Gln Met Leu Thr Ala210 215 220 Pro Ala Ala Gln Lys Ala Val Ala Asp Thr Thr Ser Val Ala ThrSer 225 230 235 240 Asn Gly Leu Ser Tyr Ala Pro Asn His Ala Tyr Asn ProMet Asn Ala 245 250 255 Gly Leu Gln Pro Gln Thr Ala Ala Phe Lys Glu GluVal Ala Ser Ala 260 265 270 Phe Gly Ile Thr Ser Phe Ser Gly Tyr Arg ProGly Asp Pro Gly Asp 275 280 285 His Gly Lys Gly Leu Ala Ile Asp Phe MetVal Pro Val Ser Ser Thr 290 295 300 Leu Gly Asp Gln Val Ala Gln Tyr AlaIle Asp His Met Ala Glu Arg 305 310 315 320 Gly Ile Ser Tyr Val Ile TrpLys Gln Arg Phe Tyr Ala Pro Phe Ala 325 330 335 Ser Ile Tyr Gly Pro AlaTyr Thr Trp Asn Pro Met Pro Asp Arg Gly 340 345 350 Ser Ile Thr Glu AsnHis Tyr Asp His Val His Val Ser Phe Asn Ala 355 360 365 13 1095 DNA S.pyogenes 13 caagagtgga caccacgatc ggttacagaa atcaagtctg aactcgtcctagttgataat 60 gtttttactt atactgtaaa atacggtgac actttaagca caattgctgaagcaatgggg 120 attgatgtgc atgtcttagg agatattaat catattgcta atattgacctaatttttcca 180 gacacgatcc taacagcaaa ctacaatcaa cacggtcagg caacgaatttgacggttcaa 240 gcacctgctt ctagtccagc tagcgttagt catgtaccta gcagtgagccattaccccaa 300 gcatctgcca cctctcaacc gactgttcct atggcaccac ctgcgacaccatctgatgtc 360 ccaacgacac cattcgcatc tgcaaagcca gatagttctg tgacagcgtcatctgagctc 420 acatcgtcaa cgaatgatgt ttcgactgag ttgtctagcg aatcacaaaagcagccagaa 480 gtaccacaag aagcagttcc aactcctaaa gcagctgaaa cgactgaagtcgaacctaag 540 acagacatct cagaagcccc aacttcagct aataggcctg tacctaacgagagtgcttca 600 gaagaagttt cttctgcggc cccagcacaa gccccagcag aaaaagaagaaacctctgcg 660 ccagcagcac aaaaagctgt agctgacacc acaagtgttg caacctcaaatggcctttct 720 tacgctccaa accatgccta caatccaatg aatgcagggc ttcaaccacaaacagcagcc 780 ttcaaagaag aagtggcttc tgcctttggt attacgtcat ttagtggttaccgtccaggt 840 gatccaggag atcatggtaa aggtttggcc attgatttta tggtgcctgaaaattctgct 900 cttggtgatc aagttgctca atatgccatt gaccatatgg cagagcgtggtatttcatac 960 gttatttgga aacagcgatt ctatgcgcca tttgcaagta tttacggaccagcctacaca 1020 tggaacccca tgccagatcg cggcagtatt acagaaaacc attatgatcatgttcatgtc 1080 tcctttaatg cttaa 1095 14 364 PRT S. pyogenes 14 Gln GluTrp Thr Pro Arg Ser Val Thr Glu Ile Lys Ser Glu Leu Val 1 5 10 15 LeuVal Asp Asn Val Phe Thr Tyr Thr Val Lys Tyr Gly Asp Thr Leu 20 25 30 SerThr Ile Ala Glu Ala Met Gly Ile Asp Val His Val Leu Gly Asp 35 40 45 IleAsn His Ile Ala Asn Ile Asp Leu Ile Phe Pro Asp Thr Ile Leu 50 55 60 ThrAla Asn Tyr Asn Gln His Gly Gln Ala Thr Asn Leu Thr Val Gln 65 70 75 80Ala Pro Ala Ser Ser Pro Ala Ser Val Ser His Val Pro Ser Ser Glu 85 90 95Pro Leu Pro Gln Ala Ser Ala Thr Ser Gln Pro Thr Val Pro Met Ala 100 105110 Pro Pro Ala Thr Pro Ser Asp Val Pro Thr Thr Pro Phe Ala Ser Ala 115120 125 Lys Pro Asp Ser Ser Val Thr Ala Ser Ser Glu Leu Thr Ser Ser Thr130 135 140 Asn Asp Val Ser Thr Glu Leu Ser Ser Glu Ser Gln Lys Gln ProGlu 145 150 155 160 Val Pro Gln Glu Ala Val Pro Thr Pro Lys Ala Ala GluThr Thr Glu 165 170 175 Val Glu Pro Lys Thr Asp Ile Ser Glu Ala Pro ThrSer Ala Asn Arg 180 185 190 Pro Val Pro Asn Glu Ser Ala Ser Glu Glu ValSer Ser Ala Ala Pro 195 200 205 Ala Gln Ala Pro Ala Glu Lys Glu Glu ThrSer Ala Pro Ala Ala Gln 210 215 220 Lys Ala Val Ala Asp Thr Thr Ser ValAla Thr Ser Asn Gly Leu Ser 225 230 235 240 Tyr Ala Pro Asn His Ala TyrAsn Pro Met Asn Ala Gly Leu Gln Pro 245 250 255 Gln Thr Ala Ala Phe LysGlu Glu Val Ala Ser Ala Phe Gly Ile Thr 260 265 270 Ser Phe Ser Gly TyrArg Pro Gly Asp Pro Gly Asp His Gly Lys Gly 275 280 285 Leu Ala Ile AspPhe Met Val Pro Glu Asn Ser Ala Leu Gly Asp Gln 290 295 300 Val Ala GlnTyr Ala Ile Asp His Met Ala Glu Arg Gly Ile Ser Tyr 305 310 315 320 ValIle Trp Lys Gln Arg Phe Tyr Ala Pro Phe Ala Ser Ile Tyr Gly 325 330 335Pro Ala Tyr Thr Trp Asn Pro Met Pro Asp Arg Gly Ser Ile Thr Glu 340 345350 Asn His Tyr Asp His Val His Val Ser Phe Asn Ala 355 360 15 1074 DNAS. pyogenes 15 caagagtgga caccacgatc ggttacagaa atcaagtctg aactcgtcctagttgataat 60 gtttttactt atacagtaaa atacggtgac actttaagca caattgctgaagcaatgggg 120 attgatgtgc atgtcttagg agatattaat catattgcta atattgacttaatttttcca 180 gacacgatcc taacagcaaa ctacaatcaa cacggtcagg caacgactttgacggttcaa 240 gcacctgctt ctagtccagc tagcgttagt catgtaccta gcagtgagccattaccccaa 300 gcatctgcca cctctcaacc gactgttcct atggcaccat ctgcgacaccattagcatct 360 gcaaagccag atagttctgt gacagcgtca tctgagctca catcgtcaacgaatgatgtt 420 tcgactgagt cgtctagcga atcacaaaag cagccagaag taccacaagaagcagttcca 480 actcctaaag cagctgaaac gactgaagtc gaacctaaga cagacatctcagaagaccca 540 acttcagcta ataggcctgt acctaacgag agtgcttcag aagaagtttcttctgcggcc 600 ccagcacaag ccccagcaga aaaagaagaa acctctgcgc cagcagcacaaaaagctgta 660 gctgacacca caagtgttgc aacctcaaac ggcctttctt acgctccaaaccatgcctac 720 aatccaatga atgcagggct tcaaccacaa acagcagcct tcaaagaagaagtggcttct 780 gcctttggta ttacgtcatt tagtggttac cgtccaggtg acccaggagatcatggtaaa 840 ggtttggcca ttgattttat ggtgcctgaa aattctgctc ttggtgatcaagttgctcaa 900 tatgccattg accatatggc agagcgtggt atttcatacg ttatttggaaacagcgattc 960 tatgcgccat ttgcaagtat ttacggacca gcttacacat ggaaccccatgccagatcgc 1020 ggcagtatta cagaaaacca ttatgatcat gttcatgtct cctttaatgcttaa 1074 16 357 PRT S. pyogenes 16 Gln Glu Trp Thr Pro Arg Ser Val ThrGlu Ile Lys Ser Glu Leu Val 1 5 10 15 Leu Val Asp Asn Val Phe Thr TyrThr Val Lys Tyr Gly Asp Thr Leu 20 25 30 Ser Thr Ile Ala Glu Ala Met GlyIle Asp Val His Val Leu Gly Asp 35 40 45 Ile Asn His Ile Ala Asn Ile AspLeu Ile Phe Pro Asp Thr Ile Leu 50 55 60 Thr Ala Asn Tyr Asn Gln His GlyGln Ala Thr Thr Leu Thr Val Gln 65 70 75 80 Ala Pro Ala Ser Ser Pro AlaSer Val Ser His Val Pro Ser Ser Glu 85 90 95 Pro Leu Pro Gln Ala Ser AlaThr Ser Gln Pro Thr Val Pro Met Ala 100 105 110 Pro Ser Ala Thr Pro LeuAla Ser Ala Lys Pro Asp Ser Ser Val Thr 115 120 125 Ala Ser Ser Glu LeuThr Ser Ser Thr Asn Asp Val Ser Thr Glu Ser 130 135 140 Ser Ser Glu SerGln Lys Gln Pro Glu Val Pro Gln Glu Ala Val Pro 145 150 155 160 Thr ProLys Ala Ala Glu Thr Thr Glu Val Glu Pro Lys Thr Asp Ile 165 170 175 SerGlu Asp Pro Thr Ser Ala Asn Arg Pro Val Pro Asn Glu Ser Ala 180 185 190Ser Glu Glu Val Ser Ser Ala Ala Pro Ala Gln Ala Pro Ala Glu Lys 195 200205 Glu Glu Thr Ser Ala Pro Ala Ala Gln Lys Ala Val Ala Asp Thr Thr 210215 220 Ser Val Ala Thr Ser Asn Gly Leu Ser Tyr Ala Pro Asn His Ala Tyr225 230 235 240 Asn Pro Met Asn Ala Gly Leu Gln Pro Gln Thr Ala Ala PheLys Glu 245 250 255 Glu Val Ala Ser Ala Phe Gly Ile Thr Ser Phe Ser GlyTyr Arg Pro 260 265 270 Gly Asp Pro Gly Asp His Gly Lys Gly Leu Ala IleAsp Phe Met Val 275 280 285 Pro Glu Asn Ser Ala Leu Gly Asp Gln Val AlaGln Tyr Ala Ile Asp 290 295 300 His Met Ala Glu Arg Gly Ile Ser Tyr ValIle Trp Lys Gln Arg Phe 305 310 315 320 Tyr Ala Pro Phe Ala Ser Ile TyrGly Pro Ala Tyr Thr Trp Asn Pro 325 330 335 Met Pro Asp Arg Gly Ser IleThr Glu Asn His Tyr Asp His Val His 340 345 350 Val Ser Phe Asn Ala 35517 1113 DNA S. pneumonia 17 atgaagaaaa gaatgttatt agcgtcaaca gtagccttgtcatttgcccc agtattggca 60 actcaagcag aagaagttct ttggactgca cgtagtgttgagcaaatcca aaacgatttg 120 actaaaacgg acaacaaaac aagttatacc gtacagtatggtgatacttt gagcaccatt 180 gcagaagcct tgggtgtaga tgtcacagtg cttgcgaatctgaacaaaat cactaatatg 240 gacttgattt tcccagaaac tgttttgaca acgactgtcaatgaagcaga agaagtaaca 300 gaagttgaaa tccaaacacc tcaagcagac tctagtgaagaagtgacaac tgcgacagca 360 gatttgacca ctaatcaagt gaccgttgat gatcaaactgttcaggttgc agacctttct 420 caaccaattg cagaagttac aaagacagtg attgcttctgaagaagtggc accatctacg 480 ggcacttctg tcccagagga gcaaacgacc gaaacaactcgcccagttga agaagcaact 540 cctcaggaaa cgactccagc tgagaagcag gaaacacaagcaagccctca agctgcatca 600 gcagtggaag taactacaac aagttcagaa gcaaaagaagtagcatcatc aaatggagct 660 acagcagcag tttctactta tcaaccagaa gagacgaaaataatttcaac aacttacgag 720 gctccagctg cgcccgatta tgctggactt gcagtagcaaaatctgaaaa tgcaggtctt 780 caaccacaaa cagctgcctt taaagaagaa attgctaacttgtttggcat tacatccttt 840 agtggttatc gtccaggaga cagtggagat cacggaaaaggtttggctat cgactttatg 900 gtaccagaac gttcagaatt aggggataag attgcggaatatgctattca aaatatggcc 960 agccgtggca ttagttacat catctggaaa caacgtttctatgctccatt cgatagcaaa 1020 tatgggccag ctaacacttg gaacccaatg ccagaccgtggtagtgtgac agaaaatcac 1080 tatgatcacg ttcacgtttc aatgaatgga taa 1113 18370 PRT S. pneumonia 18 Met Lys Lys Arg Met Leu Leu Ala Ser Thr Val AlaLeu Ser Phe Ala 1 5 10 15 Pro Val Leu Ala Thr Gln Ala Glu Glu Val LeuTrp Thr Ala Arg Ser 20 25 30 Val Glu Gln Ile Gln Asn Asp Leu Thr Lys ThrAsp Asn Lys Thr Ser 35 40 45 Tyr Thr Val Gln Tyr Gly Asp Thr Leu Ser ThrIle Ala Glu Ala Leu 50 55 60 Gly Val Asp Val Thr Val Leu Ala Asn Leu AsnLys Ile Thr Asn Met 65 70 75 80 Asp Leu Ile Phe Pro Glu Thr Val Leu ThrThr Thr Val Asn Glu Ala 85 90 95 Glu Glu Val Thr Glu Val Glu Ile Gln ThrPro Gln Ala Asp Ser Ser 100 105 110 Glu Glu Val Thr Thr Ala Thr Ala AspLeu Thr Thr Asn Gln Val Thr 115 120 125 Val Asp Asp Gln Thr Val Gln ValAla Asp Leu Ser Gln Pro Ile Ala 130 135 140 Glu Val Thr Lys Thr Val IleAla Ser Glu Glu Val Ala Pro Ser Thr 145 150 155 160 Gly Thr Ser Val ProGlu Glu Gln Thr Thr Glu Thr Thr Arg Pro Val 165 170 175 Glu Glu Ala ThrPro Gln Glu Thr Thr Pro Ala Glu Lys Gln Glu Thr 180 185 190 Gln Ala SerPro Gln Ala Ala Ser Ala Val Glu Val Thr Thr Thr Ser 195 200 205 Ser GluAla Lys Glu Val Ala Ser Ser Asn Gly Ala Thr Ala Ala Val 210 215 220 SerThr Tyr Gln Pro Glu Glu Thr Lys Ile Ile Ser Thr Thr Tyr Glu 225 230 235240 Ala Pro Ala Ala Pro Asp Tyr Ala Gly Leu Ala Val Ala Lys Ser Glu 245250 255 Asn Ala Gly Leu Gln Pro Gln Thr Ala Ala Phe Lys Glu Glu Ile Ala260 265 270 Asn Leu Phe Gly Ile Thr Ser Phe Ser Gly Tyr Arg Pro Gly AspSer 275 280 285 Gly Asp His Gly Lys Gly Leu Ala Ile Asp Phe Met Val ProGlu Arg 290 295 300 Ser Glu Leu Gly Asp Lys Ile Ala Glu Tyr Ala Ile GlnAsn Met Ala 305 310 315 320 Ser Arg Gly Ile Ser Tyr Ile Ile Trp Lys GlnArg Phe Tyr Ala Pro 325 330 335 Phe Asp Ser Lys Tyr Gly Pro Ala Asn ThrTrp Asn Pro Met Pro Asp 340 345 350 Arg Gly Ser Val Thr Glu Asn His TyrAsp His Val His Val Ser Met 355 360 365 Asn Gly 370 19 1183 DNA S.pyogenes misc_difference (428)...(448) nnnnnnnnnnnnnnnnnnnnn can bectgatgtccaacgacaccat or absent 19 atgattatta ctaaaaagag yttatttgtgacaagtgtcg ctttgtcgtt agyacctttg 60 gcgacagcrc aggcacaaga gtggacaccacgatcggtta casaaatcaa gtctgaactc 120 gtcctagttg ataatgtttt tacttataywgtaaaatacg gtgacacttt aagcacaatt 180 gctgaagcaa tgggrattga tgtgcatgtcttaggagata ttaatcatat tgctaatatt 240 gacytaattt ttccagacac gatcctaacagcmaactaca aycaacacgg tcaggcaacg 300 amtttgacgg ttcaagcrcc tgcttctagtccakctagcg ttagtcatgt acctagcagt 360 gagccattac cccaagcatc tgccacctctcaaycgactr ttcctatggc accayctgcg 420 acaccatnnn nnnnnnnnnn nnnnnnnntmgcatctgcaa agccagatag ttytgtgaca 480 gcgtcatctg agctcacatc rtcaacgaatgatgtttcga ctgagtygtc tagcgaatca 540 caaaagcagc cagaagtacc acaagaagcagwwccaactc ctaaagcagc tgaamssact 600 gaagtcgaac ctaagacaga catctcagargmyycaactt cagctaatag gcctgtacct 660 aacgrragtg cttcagaaga agyttcttctgcggccccag cacaagcycc agcagaaaaa 720 gaagaaacct ctnnnnnnnn nnnngcgccagcagcacaaa aagctgtagc tgacaccaca 780 agtgttgcaa cctcaaaygg cctttcttacgctccaaacc atgcctacaa tccaatgaat 840 gcagggcttc aaccacaaac agcagccttcaaagaagaag tgncttctgc ctttggtatt 900 acgtcattta gtggttaccg tccaggwgayccaggagatc atnggtaaag gwttrgccat 960 tgaytttatg gtrcckgwwa rytctrckcttggtgatcaa gttgctcaat atgccattga 1020 ccatatggca gassgtggta tttcatacgttatttggaaa cagcgattct atgcgccatt 1080 tgcaagtatt tacggaccag cytacacatggaaccccatg ccagatcgcg gcagtattac 1140 agwwwwccat tatgatcatg ttcatgtctcctttaatgct taa 1183 20 393 PRT s. pyogenes VARIANT (18)...(18) Xaa = Alaor Val 20 Met Ile Ile Thr Lys Lys Ser Leu Phe Val Thr Ser Val Ala LeuSer 1 5 10 15 Leu Xaa Pro Leu Ala Thr Ala Gln Ala Gln Glu Trp Thr ProArg Ser 20 25 30 Val Thr Glx Ile Lys Ser Glu Leu Val Leu Val Asp Asn ValPhe Thr 35 40 45 Tyr Xaa Val Lys Tyr Gly Asp Thr Leu Ser Thr Ile Ala GluAla Met 50 55 60 Gly Ile Asp Val His Val Leu Gly Asp Ile Asn His Ile AlaAsn Ile 65 70 75 80 Asp Leu Ile Phe Pro Asp Thr Ile Leu Thr Ala Asn TyrAsn Gln His 85 90 95 Gly Gln Ala Thr Xaa Leu Thr Val Gln Ala Pro Ala SerSer Pro Xaa 100 105 110 Ser Val Ser His Val Pro Ser Ser Glu Pro Leu ProGln Ala Ser Ala 115 120 125 Thr Ser Gln Xaa Thr Xaa Pro Met Ala Pro XaaAla Thr Pro Xaa Xaa 130 135 140 Xaa Xaa Xaa Xaa Xaa Xaa Ala Ser Ala LysPro Asp Ser Xaa Val Thr 145 150 155 160 Ala Ser Ser Glu Leu Thr Ser SerThr Asn Asp Val Ser Thr Glu Xaa 165 170 175 Ser Ser Glu Ser Gln Lys GlnPro Glu Val Pro Gln Glu Ala Xaa Pro 180 185 190 Thr Pro Lys Ala Ala GluXaa Thr Glu Val Glu Pro Lys Thr Asp Ile 195 200 205 Ser Glu Xaa Xaa ThrSer Ala Asn Arg Pro Val Pro Asn Xaa Ser Ala 210 215 220 Ser Glu Glu XaaSer Ser Ala Ala Pro Ala Gln Ala Pro Ala Glu Lys 225 230 235 240 Glu XaaXaa Xaa Xaa Xaa Xaa Xaa Ala Pro Ala Ala Gln Lys Ala Val 245 250 255 AlaAsp Thr Thr Ser Val Ala Thr Ser Asn Gly Leu Ser Tyr Ala Pro 260 265 270Asn His Ala Tyr Asn Pro Met Asn Ala Gly Leu Gln Pro Gln Thr Ala 275 280285 Ala Phe Lys Glu Glu Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Lys Gly Leu Ala Ile305 310 315 320 Asp Phe Met Val Pro Xaa Xaa Ser Xaa Leu Gly Asp Gln ValAla Gln 325 330 335 Tyr Ala Ile Asp His Met Ala Xaa Xaa Gly Ile Ser TyrVal Ile Trp 340 345 350 Lys Gln Arg Phe Tyr Ala Pro Phe Ala Ser Ile TyrGly Pro Ala Tyr 355 360 365 Thr Trp Asn Pro Met Pro Asp Arg Gly Ser IleThr Xaa Xaa His Tyr 370 375 380 Asp His Val His Val Ser Phe Asn Ala 385390 21 32 DNA Artificial Sequence DMAR16 Oligonucleotide 21 caggccatggagtggacacc acgatcggtt ac 32 22 37 DNA Artificial Sequence DMAR17Oligonucleotide 22 gccgctcgag agcattaaag gagacatgaa catgatc 37 23 25 PRTArtificial Sequence Signal peptide predicted from analysis of SEQ ID NO223 Met Ile Ile Thr Lys Lys Ser Leu Phe Val Thr Ser Val Ala Leu Ser 1 510 15 Leu Ala Pro Leu Ala Thr Ala Gln Ala 20 25 24 5 PRT ArtificialSequence VARIANT (3)...(3) Xaa = Any Amino Acid 24 Leu Pro Xaa Thr Gly 15 25 6 PRT Artificial Sequence IgA binding motif 25 Met Leu Lys Lys IleGlu 1 5 26 28 DNA Artificial Sequence DMAR69 oligonucleotide 26ctgggaagat tatctagcac attaatac 28 27 25 DNA Artificial Sequence DMAR72oligonucleotide 27 cataacgtta aaactgtcta aaggg 25 28 34 DNA ArtificialSequence DMAR24 oligonucleotide 28 tacccggatc cccaagagtg gacaccacga tcgg34 29 36 DNA Artificial Sequence DMAR25 oligonucleotide 29 gcgctcgtcgacgcgtatct cagcctctta tagggc 36

What is claimed is:
 1. An isolated polynucleotide encoding a polypeptidehaving at least 70% identity to a second polypeptide having a sequencechosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments,analogues or derivatives thereof.
 2. A polynucleotide according to claim1, wherein said polynucleotide encodes a polypeptide having at least 95%identity to the second polypeptide.
 3. An isolated polynucleotideencoding a polypeptide having at least 70% identity to a secondpolypeptide having a sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16 or
 20. 4. A polynucleotide according to claim 3, wherein saidpolynucleotide encodes a polypeptide having at least 95% identity to thesecond polypeptide.
 5. An isolated polynucleotide encoding a polypeptidecapable of generating antibodies having binding specificity for apolypeptide having a sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 20 or fragments, analogues or derivatives thereof.
 6. Anisolated polynucleotide encoding a polypeptide capable of generatingantibodies having binding specificity for a polypeptide having asequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or
 20. 7.An isolated polynucleotide that is complementary to the polynucleotideof any of claims 1 to
 6. 8. The polynucleotide of any of claims 1 to 6,wherein said polynucleotide is DNA.
 9. The polynucleotide of any ofclaims 1 to 6, wherein said polynucleotide is RNA.
 10. A polynucleotidewhich hybridizes under stringent conditions to a second polynucleotidehaving a sequence chosen from: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,19.
 11. A polynucleotide according to claim 10 wherein saidpolynucleotide has at least 95% complementarity to the secondpolynucleotide.
 12. A polynucleotide which hybridizes under stringentconditions to a second polynucleotide having a sequence chosen from: SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
 19. 13. A polynucleotide according toclaim 12 wherein said polynucleotide has at least 95% complementarity tothe second polynucleotide.
 14. An isolated polynucleotide having asequence comprising SEQ ID NO:19.
 15. A vector comprising thepolynucleotide of any of claims 1 to 6 or 10 to 14, wherein said DNA isoperably linked to an expression control region.
 16. A vector comprisingthe polynucleotide of claim 7, wherein said DNA is operably linked to anexpression control region.
 17. A host cell transfected with the vectorof claim
 15. 18. A host cell transfected with the vector of claim 16.19. A process for producing a polypeptide comprising culturing a hostcell according to claim 17 under conditions suitable for expression ofsaid polypeptide.
 20. A process for producing a polypeptide comprisingculturing a host cell according to claim 18 under condition suitable forexpression of said polypeptide.
 21. An isolated polypeptide having atleast 70% identity to a second polypeptide having an amino acid sequencechosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments,analogues or derivatives thereof.
 22. An isolated polypeptide having atleast 95% identity to a second polypeptide having an amino acid sequencechosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 20 or fragments,analogues or derivatives thereof.
 23. An isolated polypeptide having atleast 70% identity to a second polypeptide having an amino acid sequencechosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or
 20. 24. Anisolated polypeptide having at least 95% identity to a secondpolypeptide having an amino acid sequence chosen from: SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16 or
 20. 25. An isolated polypeptide capable ofgenerating antibodies having binding specificity for a secondpolypeptide having a sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 20 or fragments, analogues or derivatives thereof.
 26. Anisolated polypeptide capable of generating antibodies having bindingspecificity for a second polypeptide having a sequence chosen from: SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or
 20. 27. An isolated polypeptidehaving an amino acid sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 20 or fragments, analogues or derivatives thereof.
 28. Anisolated polypeptide having an amino acid sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16 or
 20. 29. An isolated polypeptideaccording to any of claims 21 to 28, wherein the N-terminal Met residueis deleted.
 30. An isolated polypeptide according to any of claims 21 to28, wherein the secretory amino acid sequence is deleted.
 31. Anisolated polypeptide according to claim 29, wherein the secretory aminoacid sequence is deleted.
 32. A vaccine composition comprising apolypeptide according to any one of claims 21 to 28 and apharmaceutically acceptable carrier, diluent or adjuvant.
 33. A vaccinecomposition comprising a polypeptide according to claim 29 and apharmaceutically acceptable carrier, diluent or adjuvant.
 34. A vaccinecomposition comprising a polypeptide according to claim 30 and apharmaceutically acceptable carrier, diluent or adjuvant.
 35. A vaccinecomposition comprising a polypeptide according to claim 31 and apharmaceutically acceptable carrier, diluent or adjuvant.
 36. A methodfor therapeutic or prophylactic treatment of pharyngitis, erysipelas andimpetigo, scarlet fever, and invasive diseases such as bacteremia andnecrotizing fasciitis in an individual susceptible to pharyngitis,erysipelas and impetigo, scarlet fever, and invasive diseases such asbacteremia and necrotizing fasciitis and also toxic shock comprisingadministering to said individual a therapeutic or prophylactic amount ofa composition according to claim
 32. 37. A method for therapeutic orprophylactic treatment of pharyngitis, erysipelas and impetigo, scarletfever, and invasive diseases such as bacteremia and necrotizingfasciitis in an individual susceptible to pharyngitis, erysipelas andimpetigo, scarlet fever, and invasive diseases such as bacteremia andnecrotizing fasciitis and also toxic shock comprising administering tosaid individual a therapeutic or prophylactic amount of a compositionaccording to claim
 33. 38. A method for therapeutic or prophylactictreatment of pharyngitis, erysipelas and impetigo, scarlet fever, andinvasive diseases such as bacteremia and necrotizing fasciitis in anindividual susceptible to pharyngitis, erysipelas and impetigo, scarletfever, and invasive diseases such as bacteremia and necrotizingfasciitis and also toxic shock comprising administering to saidindividual a therapeutic or prophylactic amount of a compositionaccording to claim
 34. 39. A method for therapeutic or prophylactictreatment of pharyngitis, erysipelas and impetigo, scarlet fever, andinvasive diseases such as bacteremia and necrotizing fasciitis in anindividual susceptible to pharyngitis, erysipelas and impetigo, scarletfever, and invasive diseases such as bacteremia and necrotizingfasciitis and also toxic shock comprising administering to saidindividual a therapeutic or prophylactic amount of a compositionaccording to claim
 35. 40. A method for therapeutic or prophylactictreatment of Streptococcus pyogenes bacterial infection in an individualsusceptible to Streptococcus pyogenes infection comprising administeringto said individual a therapeutic or prophylactic amount of a compositionaccording to claim
 32. 41. A method for therapeutic or prophylactictreatment of Streptococcus pyogenes bacterial infection in an individualsusceptible to Streptococcus pyogenes infection comprising administeringto said individual a therapeutic or prophylactic amount of a compositionaccording to claim
 33. 42. A method for therapeutic or prophylactictreatment of Streptococcus pyogenes bacterial infection in an individualsusceptible to Streptococcus pyogenes infection comprising administeringto said individual a therapeutic or prophylactic amount of a compositionaccording to claim
 34. 43. A method for therapeutic or prophylactictreatment of Streptococcus pyogenes bacterial infection in an individualsusceptible to Streptococcus pyogenes infection comprising administeringto said individual a therapeutic or prophylactic amount of a compositionaccording to claim
 35. 44. Use of a vaccine composition according toclaim 32 for the prophylactic or therapeutic treatment of Streptococcalinfection in an individual susceptible to or infected with streptococcalinfection comprising administering to said individual a prophylactic ortherapeutic amount of the composition.
 45. Use of a vaccine compositionaccording to claim 33 for the prophylactic or therapeutic treatment ofStreptococcal infection in an individual susceptible to or infected withstreptococcal infection comprising administering to said individual aprophylactic or therapeutic amount of the composition.
 46. Use of avaccine composition according to claim 34 for the prophylactic ortherapeutic treatment of Streptococcal infection in an individualsusceptible to or infected with streptococcal infection comprisingadministering to said individual a prophylactic or therapeutic amount ofthe composition.
 47. Use of a vaccine composition according to claim 35for the prophylactic or therapeutic treatment of Streptococcal infectionin an individual susceptible to or infected with streptococcal infectioncomprising administering to said individual a prophylactic ortherapeutic amount of the composition.